Science.gov

Sample records for actinic keratoses ak

  1. Actinic Keratoses

    PubMed Central

    Brown, Marc D.

    2009-01-01

    Actinic keratoses are common intra-epidermal neoplasms that lie on a continuum with squamous cell carcinoma. Tightly linked to ultraviolet irradiation, they occur in areas of chronic sun exposure, and early treatment of these lesions may prevent their progression to invasive disease. A large variety of effective treatment modalities exist, and the optimal therapeutic choice is dependent on a variety of patient- and physician-associated variables. Many established and more recent approaches are discussed in this review with a focus on efficacy and administration techniques. Several previously experimental options, such as imiquimod and photodynamic therapy, have become incorporated as first-line options for the treatment of actinic keratoses, while combination treatment strategies have been gaining in popularity. The goal of all therapies is to ultimately limit the morbidity and mortality of squamous cell carcinoma. (J Clin Aesthetic Dermatol. 2009;2(7):43–48.) PMID:20729970

  2. Handheld dual fluorescence and reflection spectroscopy system for monitoring topical low dose ALA-PDT of actinic keratoses (AK)

    NASA Astrophysics Data System (ADS)

    Charamisinau, Ivan; Keymel, Kenneth; Potter, William; Oseroff, Allan R.

    2006-02-01

    measurements and calculations, and writes data to a database. The system is currently used in a clinical trial to find the relationship between skin pigmentation, oxygen saturation in blood, photobleaching rate and optimal fluence rate for skin cancer treatment of actinic keratoses.

  3. Use of Photodynamic Therapy for Treatment of Actinic Keratoses in Organ Transplant Recipients

    PubMed Central

    Wlodek, Christina; Ali, Faisal R.; Lear, John T.

    2013-01-01

    Solid organ transplant recipients are predisposed to actinic keratoses (AK) and nonmelanoma skin cancers, owing to the lifelong immunosuppression required. Today, increasing numbers of organ transplants are being performed and organ transplant recipients (OTRs) are surviving much longer. Photodynamic therapy (PDT) is proving a highly effective treatment modality for AK amongst this susceptible group of patients. Following an overview of the pathogenesis of AK amongst OTRs, the authors review current safety and efficacy data and how this relates to the role of PDT for the treatment of AK in OTRs. PMID:23509711

  4. Consensus recommendations on the use of daylight photodynamic therapy with methyl aminolevulinate cream for actinic keratoses in Australia.

    PubMed

    See, Jo-Ann; Shumack, Stephen; Murrell, Dedee F; Rubel, Diana M; Fernández-Peñas, Pablo; Salmon, Robert; Hewitt, Daniel; Foley, Peter; Spelman, Lynda

    2016-08-01

    Australia has the highest prevalence of actinic keratoses (AK) worldwide. Because of the risk of transformation of AK to invasive squamous cell carcinomas, consensus guidelines recommend that AK are removed using appropriate therapies to prevent progression to invasive disease. Daylight photodynamic therapy (PDT) is emerging as an efficacious treatment for AK, particularly for patients who require treatment of large areas of chronic actinic damage that can be exposed easily to daylight. Daylight PDT with methyl aminolevulinate (MAL) cream is a simple treatment for AK, almost painless, well tolerated and convenient, requiring minimal time in the clinic. Randomised controlled studies from northern Europe and Australia support the use of daylight PDT as an effective therapy for grade I and II AK on the face and scalp. There is sufficient daylight to conduct daylight PDT in Australia at any time of the year and during most weather conditions. Hence, daylight PDT with MAL can be included as an effective and well-tolerated new treatment option for the treatment of AK in Australia. These consensus recommendations provide guidelines for Australian clinicians on the use of daylight PDT in the treatment of diagnosed AK. PMID:26033230

  5. Consensus recommendations on the use of daylight photodynamic therapy with methyl aminolevulinate cream for actinic keratoses in Australia.

    PubMed

    See, Jo-Ann; Shumack, Stephen; Murrell, Dedee F; Rubel, Diana M; Fernández-Peñas, Pablo; Salmon, Robert; Hewitt, Daniel; Foley, Peter; Spelman, Lynda

    2016-08-01

    Australia has the highest prevalence of actinic keratoses (AK) worldwide. Because of the risk of transformation of AK to invasive squamous cell carcinomas, consensus guidelines recommend that AK are removed using appropriate therapies to prevent progression to invasive disease. Daylight photodynamic therapy (PDT) is emerging as an efficacious treatment for AK, particularly for patients who require treatment of large areas of chronic actinic damage that can be exposed easily to daylight. Daylight PDT with methyl aminolevulinate (MAL) cream is a simple treatment for AK, almost painless, well tolerated and convenient, requiring minimal time in the clinic. Randomised controlled studies from northern Europe and Australia support the use of daylight PDT as an effective therapy for grade I and II AK on the face and scalp. There is sufficient daylight to conduct daylight PDT in Australia at any time of the year and during most weather conditions. Hence, daylight PDT with MAL can be included as an effective and well-tolerated new treatment option for the treatment of AK in Australia. These consensus recommendations provide guidelines for Australian clinicians on the use of daylight PDT in the treatment of diagnosed AK.

  6. Consensus recommendations on the use of daylight photodynamic therapy with methyl aminolevulinate cream for actinic keratoses in Australia

    PubMed Central

    Shumack, Stephen; Murrell, Dedee F; Rubel, Diana M; Fernández‐Peñas, Pablo; Salmon, Robert; Hewitt, Daniel; Foley, Peter; Spelman, Lynda

    2015-01-01

    Abstract Australia has the highest prevalence of actinic keratoses (AK) worldwide. Because of the risk of transformation of AK to invasive squamous cell carcinomas, consensus guidelines recommend that AK are removed using appropriate therapies to prevent progression to invasive disease. Daylight photodynamic therapy (PDT) is emerging as an efficacious treatment for AK, particularly for patients who require treatment of large areas of chronic actinic damage that can be exposed easily to daylight. Daylight PDT with methyl aminolevulinate (MAL) cream is a simple treatment for AK, almost painless, well tolerated and convenient, requiring minimal time in the clinic. Randomised controlled studies from northern Europe and Australia support the use of daylight PDT as an effective therapy for grade I and II AK on the face and scalp. There is sufficient daylight to conduct daylight PDT in Australia at any time of the year and during most weather conditions. Hence, daylight PDT with MAL can be included as an effective and well‐tolerated new treatment option for the treatment of AK in Australia. These consensus recommendations provide guidelines for Australian clinicians on the use of daylight PDT in the treatment of diagnosed AK. PMID:26033230

  7. Effective Combination of Photodynamic Therapy and Imiquimod 5% Cream in the Treatment of Actinic Keratoses: Three Cases

    PubMed Central

    Held, Laura; Eigentler, Thomas Kurt; Leiter, Ulrike; Garbe, Claus; Berneburg, Mark-Jürgen

    2013-01-01

    Background. The therapy for actinic keratoses includes photodynamic therapy (PDT) and imiquimod 5% cream. The sequential use of both could result in better clinical outcomes. Objectives. To enhance efficacy of therapies while improving tolerability, convenience, and patient adherence with a scheme combining two concomitant or sequential AK treatments. Methods. All patients underwent one session of conventional PDT. Two weeks after, the PDT imiquimod 5% cream was applied to the treatment area once daily for three days per week. One course continued for four weeks followed by a clinical evaluation and decision about further treatment. Patients who had not cleared all of their AK lesions in the treatment area in course 1 participated in a second 4-week course of treatment. Limitations. Small size of population. Results. Three participants were enrolled. Two patients showed complete clinical clearance of AKs. The effect was also noted after long-term followup, at months seven and eleven. No subject discontinued for an adverse event. There were severe local skin reactions in two participants which were severe erythema, scaling, and crusting. One patient showed no response to the therapy. Conclusions. Photodynamic therapy followed by imiquimod was well tolerated and improved reduction of actinic keratoses. This initial proof-of-concept should be studied in larger clinical trials. PMID:23484071

  8. The Incidence of Nonmelanoma Skin Cancers and Actinic Keratoses in South Florida

    PubMed Central

    Zarraga, Matthew B.

    2012-01-01

    Background: Incidence of nonmelanoma skin cancer and actinic keratoses appears to be increasing worldwide due to increasing levels of ultraviolet radiation, lifestyle changes, and an aging population. Because of its demographics and geographic location, the population of South Florida is at risk for high rates of nonmelanoma skin cancer and actinic keratoses. Objective: To determine the incidence of nonmelanoma skin cancer and actinic keratoses in two populations in South Florida by measuring treatments by dermatologists in health maintenance organization gatekeeper populations. Methods: The incidence of nonmelanoma skin cancer and actinic keratoses in South Florida was determined by evaluating the number of nonmelanoma skin cancers and actinic keratoses treated by dermatologists (Current Procedural Terminology [CPT] Code Analysis) in two health maintenance organization populations; “commercial” (age 0–65, mean 27) and Medicare (age 65+, mean 68) in the calendar year 1996. Results: The incidence of treatment of nonmelanoma skin cancer was 466.5 per 100,000 people per year in the “commercial” (age 0 to 65) population and 10,689.8 per 100,000 people per year in the Medicare age population. The incidence of treated actinic keratoses was 4,464.6 per 100,000 people per year and 110,450.3 in each population respectively. Conclusion: The studied populations in South Florida appear to have some of the highest incidence rates of nonmelanoma skin cancer in the world and extremely high rates of actinic keratoses. The findings suggest that there is an epidemic of nonmelanoma skin cancer in the South Florida community, which has significant implications for the future medical needs of both “commercial” and Medicare-age populations. PMID:22708003

  9. Clinical, dermoscopic and immunohistochemical assessment of actinic keratoses and evaluation of the effectiveness of diclofenac therapy with immunohistochemical analysis.

    PubMed

    Çayirli, Mutlu; Köse, Osman; Demiriz, Murat

    2013-07-01

    Actinic keratoses (AKs) is a keratinocytic neoplasm that typically develops on the face of elderly patients. Little is known regarding the clinical, dermatoscopic and immunohistochemical assessments of AK using topical diclofenac therapy. We sought to determine these assessments and evaluate the efficacy of topical diclofenac gel in AK. In this prospective, open-label study, 44 patients with 66 AKs were treated for 12 weeks with topically applied diclofenac (3% gel in 2.5% hyaluronic acid). Immunohistopathologic analyses were performed before and after diclofenac treatment using epidermal stem cell markers such as Cytokeratin 15 (CK15), Cytokeratin 19 (CK19) and p63, in addition to proliferation markers (Bcl-2, Ki-67). Diclofenac gel was found to be effective in AK, including the hyperkeratotic type. Surprisingly, complete remission was observed at a significantly higher rate in Grade 3 lesions (p = 0.017). However, imunohistochemical and histopathologic examinations revealed that 12-week treatment periods may not be sufficient to fully cure AK. The immunohistochemical analyses revealed no change in the expression levels of CK15, CK19 and Bcl-2 following diclofenac therapy. However, the expression of Ki-67 (p = 0.042) and p63 (p = 0.030) exhibited a significant decrease after therapy. Dermatoscopy is an effective method for diagnosis of AK, and topical diclofenac sodium gel was found as an effective additional treatment modality. Since positive histopathological findings were detected in some patients even with significant remission, a 12-week treatment period should be extended even in patients presenting with positive clinical response. Importantly, anti-proliferative effects of diclofenac were demonstrated by decreased Ki-67 and p63 expression levels.

  10. Topical Colchicine Gel versus Diclofenac Sodium Gel for the Treatment of Actinic Keratoses: A Randomized, Double-Blind Study

    PubMed Central

    Faghihi, Gita; Iraji, Fariba; Behfar, Shadi; Abtahi-Naeini, Bahareh

    2016-01-01

    Introduction. Actinic keratoses (AKs), a premalignant skin lesion, are a common lesion in fair skin. Although destructive treatment remains the gold standard for AKs, medical therapies may be preferable due to the comfort and reliability .This study aims to compare the effects of topical 1% colchicine gel and 3% diclofenac sodium gel in AKs. Materials and Methods. In this randomized double-blind study, 70 lesions were selected. Patients were randomized before receiving either 1% colchicine gel or 3% diclofenac sodium cream twice a day for 6 weeks. Patients were evaluated in terms of their lesion size, treatment complications, and recurrence at 7, 30, 60, and 120 days after treatment. Results. The mean of changes in the size was significant in both groups both before and after treatment (<0.001). The mean lesion size before treatment and at 30, 60, and 120 days was not different between the two groups (p > 0.05). No case of erythema was seen in the colchicine group, while erythema was seen in 22.9% (eight cases) of patients in the diclofenac sodium group (p = 0.005). Conclusions. 1% colchicine gel was a safe and effective medication with fewer side effects and lack of recurrence of the lesion. PMID:27689135

  11. Topical Colchicine Gel versus Diclofenac Sodium Gel for the Treatment of Actinic Keratoses: A Randomized, Double-Blind Study

    PubMed Central

    Faghihi, Gita; Iraji, Fariba; Behfar, Shadi; Abtahi-Naeini, Bahareh

    2016-01-01

    Introduction. Actinic keratoses (AKs), a premalignant skin lesion, are a common lesion in fair skin. Although destructive treatment remains the gold standard for AKs, medical therapies may be preferable due to the comfort and reliability .This study aims to compare the effects of topical 1% colchicine gel and 3% diclofenac sodium gel in AKs. Materials and Methods. In this randomized double-blind study, 70 lesions were selected. Patients were randomized before receiving either 1% colchicine gel or 3% diclofenac sodium cream twice a day for 6 weeks. Patients were evaluated in terms of their lesion size, treatment complications, and recurrence at 7, 30, 60, and 120 days after treatment. Results. The mean of changes in the size was significant in both groups both before and after treatment (<0.001). The mean lesion size before treatment and at 30, 60, and 120 days was not different between the two groups (p > 0.05). No case of erythema was seen in the colchicine group, while erythema was seen in 22.9% (eight cases) of patients in the diclofenac sodium group (p = 0.005). Conclusions. 1% colchicine gel was a safe and effective medication with fewer side effects and lack of recurrence of the lesion.

  12. Topical Colchicine Gel versus Diclofenac Sodium Gel for the Treatment of Actinic Keratoses: A Randomized, Double-Blind Study.

    PubMed

    Faghihi, Gita; Elahipoor, Azam; Iraji, Fariba; Behfar, Shadi; Abtahi-Naeini, Bahareh

    2016-01-01

    Introduction. Actinic keratoses (AKs), a premalignant skin lesion, are a common lesion in fair skin. Although destructive treatment remains the gold standard for AKs, medical therapies may be preferable due to the comfort and reliability .This study aims to compare the effects of topical 1% colchicine gel and 3% diclofenac sodium gel in AKs. Materials and Methods. In this randomized double-blind study, 70 lesions were selected. Patients were randomized before receiving either 1% colchicine gel or 3% diclofenac sodium cream twice a day for 6 weeks. Patients were evaluated in terms of their lesion size, treatment complications, and recurrence at 7, 30, 60, and 120 days after treatment. Results. The mean of changes in the size was significant in both groups both before and after treatment (<0.001). The mean lesion size before treatment and at 30, 60, and 120 days was not different between the two groups (p > 0.05). No case of erythema was seen in the colchicine group, while erythema was seen in 22.9% (eight cases) of patients in the diclofenac sodium group (p = 0.005). Conclusions. 1% colchicine gel was a safe and effective medication with fewer side effects and lack of recurrence of the lesion. PMID:27689135

  13. Improved patient satisfaction using ingenol mebutate gel 0.015% for the treatment of facial actinic keratoses: a prospective pilot study

    PubMed Central

    Emilio, Joanna; Schwartz, Michelle; Feldman, Eleanor; Bieber, Amy Kalowitz; Bienenfeld, Amanda; Jung, Min-Kyung; Siegel, Daniel M; Markowitz, Orit

    2016-01-01

    Actinic keratoses (AKs), especially on areas of the face, have a negative impact on a patient’s quality of life (QoL). These lesions manifest on sun-damaged skin and have the potential to progress to squamous cell carcinoma. Field-directed therapy alone and in combination with lesion-directed treatment is effective in clearing both visible and nonvisible AK lesions. Topical treatments of AKs thus have the potential to improve a patient’s well-being. However, evidence demonstrating improvements in patient QoL is limited, and is mostly based on observational or retrospective studies. Some prospective studies have reported unchanged or even worsening QoL despite excellent treatment outcomes. Our prospective, pilot study demonstrated a significant increase in QoL in 28 subjects with AKs of the face treated with ingenol mebutate gel 0.015%. QoL was assessed at days 0 and 60 using the Skindex-16 survey. Mean overall scores improved from 24.5% at baseline to 15.5% at day 60 (P=0.031). Improvements in QoL were consistent with an 80% reduction in AK lesion number at day 60. These improved QoL findings are in line with those from a recent retrospective study using ingenol mebutate 0.015% gel. This study therefore further demonstrates the potential for field therapy to improve both treatment outcomes and patient satisfaction. PMID:27143946

  14. Topical photodynamic therapy with 5-aminolevulinic acid in the treatment of actinic keratoses: a first clinical study

    NASA Astrophysics Data System (ADS)

    Karrer, Sigrid; Szeimies, Rolf-Markus; Sauerwald, Angela; Landthaler, Michael

    1996-01-01

    In this first clinical study performed according to GCP- (good clinical practice) guidelines, efficacy, and tolerability of topical photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA) were tested in the treatment of actinic keratoses. Ten patients (6 f, 4 m) with 36 lesions (19 located on hands and arms, 17 on the head) received ALA-PDT once. Five to six hours after occlusive application of ALA (water-in-oil-emulsion containing 10% ALA) irradiation was performed with an incoherent light source. Up to 3 months after treatment patients were monitored. A score evaluating infiltration and keratosis of treated actinic keratoses allowed us to estimate therapeutic efficacy. Compared to the initial score (100%) significantly lower score-sums were observed at the 28 day follow-up at both localizations (head: 15%; hand: 67%). Complete remission (score sum 0) resulted in 71% of actinic keratoses localized on the head. Except for slight pain and burning sensations during and after irradiation there were no notable side effects. This study proved good efficacy and tolerability of topical PDT in the treatment of actinic keratoses. Whether PDT is able to compete with established treatment modalities remains to be shown in further studies.

  15. Noninvasive fluorescence monitoring of protoporphyrin IX production and clinical outcomes in actinic keratoses following short-contact application of 5-aminolevulinate

    NASA Astrophysics Data System (ADS)

    Warren, Christine B.; Lohser, Sara; Wene, Lauren C.; Pogue, Brian W.; Bailin, Philip L.; Maytin, Edward V.

    2010-09-01

    Topical 5-aminolevulinic acid (ALA) is widely used in photodynamic therapy (PDT) of actinic keratoses (AK), a type of premalignant skin lesion. However, the optimal time between ALA application and exposure to light has not been carefully investigated. Our objective is to study the kinetics of protoporphyrin IX (PpIX) accumulation in AK after short contact ALA and relate this to erythemal responses. Using a noninvasive dosimeter, PpIX fluorescence measurements (5 replicates) were taken at 20-min intervals for 2 h following ALA application, in 63 AK in 20 patients. Data were analyzed for maximal fluorescent signal obtained, kinetic slope, and changes in erythema. Our results show that PpIX accumulation was linear over time, becoming statistically higher than background in 48% of all lesions by 20 min, 92% of lesions by 1 h, and 100% of lesions by 2 h. PpIX accumulation was roughly correlated with changes in lesional erythema post-PDT. We conclude that significant amounts of PpIX are produced in all AK lesions by 2 h. The linear kinetics of accumulation suggest that shorter ALA application times may be efficacious in many patients. Noninvasive fluorescence monitoring of PpIX may be useful to delineate areas of high PpIX accumulation within precancerous areas of the skin.

  16. Real-life Data on Patient Characteristics, Cost and Effectiveness of Field-directed Treatment for Actinic Keratoses: An Observational Study.

    PubMed

    van Rijsingen, Margit C J; Seubring, Inge; Grutters, Janneke P C; Maessen-Visch, M Birgitte; Alkemade, Hans A C; van Doorn, Remco; Groenewoud, Hans; van de Kerkhof, Peter C M; van der Wilt, Gert Jan; Gerritsen, Marie-Jeanne P

    2016-03-01

    Actinic keratoses (AK) occur frequently; however, real-life clinical data on personalized treatment choice and costs are scarce. This multicentre one-year observational study investigated patient-characteristics, cost and effectiveness of methylaminolaevulinate photodynamic therapy (MAL-PDT), imiquimod (IMI) and 5-fluorour-acil (5-FU) in patients with AKs on the face/scalp. A total of 104 patients preferred MAL-PDT, 106 preferred IMI and 110 preferred 5-FU. At baseline, significant differences between treatment groups were found; most patients were severely affected (mean 32.5 AK in PDT-group, 20.2 in IMI-group, 22.8 in 5-FU-group). A mean reduction in lesions of 81% after MAL-PDT, 82% after IMI and 88% after 5-FU was found after one year. Annual costs were €1,950 for MAL-PDT, €877 for IMI and €738 for 5-FU. These results show that, compared with clinical trials, in the real-life clinical setting AK patients are usually more severely affected and treatment costs are much higher. Furthermore, patient characteristics are important factors in treatment choice.

  17. Persistence of betapapillomavirus infections as a risk factor for actinic keratoses, precursor to cutaneous squamous cell carcinoma.

    PubMed

    Plasmeijer, Elsemieke I; Neale, Rachel E; de Koning, Maurits N C; Quint, Wim G V; McBride, Penelope; Feltkamp, Mariet C W; Green, Adele C

    2009-12-01

    Human papillomaviruses from the beta genus (betaPV) are a possible cause of cutaneous squamous cell carcinoma (SCC). We assessed the extent to which betaPV infections persisted long-term in a subtropical Australian community and whether betaPV persistence is positively associated with actinic keratoses, precursor for SCC. Eyebrow hairs were collected from 171 participants of the community-based Nambour Skin Cancer Study in 1996 and 2003. Hair samples were tested for the presence of DNA from 25 different betaPV types and assessed in relation to actinic keratosis presence in 2007. In 1996, a total of 413 betaPV infections were found in 73% of participants, increasing to 490 infections among 85% in 2003. Of the total betaPV infections detected, 211 (30%) were found to persist. Age was significantly associated with betaPV persistence: those ages >60 years had 1.5-fold (95% confidence interval, 1.1-1.9) increased risk of type-specific viral persistence than those ages <40 years. After accounting for actinic keratoses at baseline, persistence of betaPV DNA resulted in a 1.4-fold (95% confidence interval, 1.0-1.9) increase in risk of having actinic keratoses on the face in 2007. In conclusion, persistent betaPV infections in this population were associated with an increased occurrence of actinic keratosis. Additional studies are needed to determine the possible association of betaPV persistence with SCC.

  18. The Effect of Multiple Sequential Light Sources to Activate Aminolevulinic Acid in the Treatment of Actinic Keratoses: A Retrospective Study

    PubMed Central

    Goldman, Mitchel P.; Fabi, Sabrina G.; Guiha, Isabella

    2014-01-01

    There is a lack of research regarding the sequential use of multiple light sources for topical 5-aminolevulinic acid activation in photodynamic therapy for actinic keratosis. This study evaluated 5-aminolevulinic acid-photodynamic therapy for actinic keratosis using blue light combined with red light, pulsed dye laser, and/or intense pulsed light in a retrospective fashion. Field-directed 5-aminolevulinic acid-photodynamic therapy was performed with blue light only, blue light + pulsed dye laser, blue light + intense pulsed light, blue light + pulsed dye laser + intense pulsed light, or blue light + red light + pulsed dye laser + intense pulsed light for nonhyperkeratotic actinic keratoses of face, scalp, or upper trunk. Blue light + intense pulsed light + pulsed dye laser produced greater patient-reported improvement in actinic keratoses than blue light or blue light + intense pulsed light and greater subject-reported improvement in overall skin quality than blue light + intense pulsed light. The addition of red light led to no further benefit in either outcome measure. Photodynamic therapy with multiple, sequential laser and light sources led to greater patient-graded improvement in actinic keratoses than that with a single light source (blue light), without significant differences in post-treatment adverse events. However, the small, widely disparate number of patients between groups and follow-up times between patients, as well as retrospective assessments based on subjective patient recall, severely limit the significance of these findings. Nevertheless, the results raise interesting questions regarding the use of multiple light and laser sources for photodynamic therapy of actinic keratoses and warrant further research with a prospective, randomized, controlled study. PMID:25276272

  19. [Procedure for daylight methyl aminolevulinate photodynamic therapy to treat actinic keratoses].

    PubMed

    Girard, C; Adamski, H; Basset-Séguin, N; Beaulieu, P; Dreno, B; Riboulet, J-L; Lacour, J-P

    2016-04-01

    Actinic keratosis (AK), also known as solar keratosis or pre-cancerous keratosis, is frequently observed in areas of skin exposed to sunlight, particularly in light-skinned patients. In France, photodynamic therapy using red light (conventional PDT) and methylamino 5-levulinate (MAL) is indicated in the treatment of thin or non-hyperkeratotic and non-pigmented multiple AK lesions or large zones covered with AK lesions. It is well-known for its efficacy but also for its side effects, especially pain during illumination, which can limit its use. An alternative to PDT using natural daylight has recently been proposed to treat actinic keratosis lesions, and results in greater flexibility as well as significant reduction in pain. The lesions are prepared as for conventional PDT, with MAL cream being applied by the physician or the patient, after which they are exposed to natural daylight for 2hours. The lesions are then gently cleansed and protected from natural light for 24hours. This paper seeks to provide a precise description of the daylight PDT procedure for the treatment of AK. PMID:27016200

  20. Apparent clinical resolution of pinnal actinic keratoses and squamous cell carcinoma in a cat using topical imiquimod 5% cream.

    PubMed

    Peters-Kennedy, Jeanine; Scott, Danny W; Miller, William H

    2008-12-01

    Imiquimod is a topical immune response modifier and stimulator used in humans to treat a number of cutaneous neoplasms. This case report describes a cat with actinic keratoses and squamous cell carcinoma of the pinnae. The pinnal lesions were treated with topical 5% imiquimod three times per week. Treatment was discontinued after 82 days of therapy. Twelve weeks of topical imiquimod application resulted in clinical resolution of the pinnal lesions. Although no post-treatment biopsies were performed, there was no relapse of the pinnal lesions in 5 months of clinical follow-up. Expected side effects were limited to erythema, crusting, alopecia, and mild discomfort at the sites of application during the first 3 weeks of application. These results suggest that topical imiquimod, although unproven, might be a therapeutic option or adjunct to therapy for cats with actinic keratoses and squamous cell carcinoma, especially those cats for whom surgery and radiation therapy are not an option.

  1. Topical AC-11 abates actinic keratoses and early squamous cell cancers in hairless mice exposed to Ultraviolet A (UVA) radiation.

    PubMed

    Mentor, Julian M; Etemadi, Amir; Patta, Abrienne M; Scheinfeld, Noah

    2015-04-01

    AC-11 is an aqueous extract of the botanical, Uncaria tomentosa, which has a variety of effects that enhance DNA repair and down regulate inflammation. AC-11 is essentially free of oxindole alkaloids (< 0.05%, w/w) but contains more than 8% carboxy alkyl esters (CAEs) as their active ingredients. Three groups of 10 outbred SK-1 hairless or SK-II hairless strains of mice each were treated with AC-11 at 0.5%, 1.5%, and 3.0% in a non-irritating, dye-free, perfume-free, and fragrance-free vanishing cream vehicle. Ten mice used vehicle only and 10 were untreated. Each concentration of AC-11 and was applied daily to the backs of the mice prior to exposure to a 1,600-watt solar simulator used in this work (Solar Light Co. Philadelphia, PA) emitting (mainly Ultraviolet A (UVA) and B (UVB) radiation) duration of the experimental period with UVB wavelengths was filtered out with a 1.0 cm Schott WG 345 filter. AC-11 with a peak absorption at 200nm does act as a sun block. We tested for and focused on clinical appearance of mice and histological appearance of tumors in mice rather than metrics of radiation generated inflammation. Tumor progression scores were assigned as follows: 4+ = extensive tumor development; 3+ = early malignancies (raised palpable plaques)(early squamous cell cancers) 2+ = firm scaling, palpable keratosis (actinic keratoses); 1+ = light scaling with erythema. Following a total cumulative dose of 738 J/cm2, 85.7% all of the irradiated control animals, which did not receive AC-11 had precancerous actinic keratosis (AK)-type lesions (2+) (64.3% versus 42.9%) or early squamous cell carcinoma (SCC) (3+) (21.4% vs. 4.8%), in comparison with 47.7 % of AC-11-treated animals. There were no significant differences between the AC-11 groups. Three months after cessation of exposure to UVA radiation, the lesions in all but three of the 14 animals which were treated with AC-11 that were still evaluable irradiated with UVA radiation progressed to papillomas and frank

  2. Topical AC-11 abates actinic keratoses and early squamous cell cancers in hairless mice exposed to Ultraviolet A (UVA) radiation.

    PubMed

    Mentor, Julian M; Etemadi, Amir; Patta, Abrienne M; Scheinfeld, Noah

    2015-04-16

    AC-11 is an aqueous extract of the botanical, Uncaria tomentosa, which has a variety of effects that enhance DNA repair and down regulate inflammation. AC-11 is essentially free of oxindole alkaloids (< 0.05%, w/w) but contains more than 8% carboxy alkyl esters (CAEs) as their active ingredients. Three groups of 10 outbred SK-1 hairless or SK-II hairless strains of mice each were treated with AC-11 at 0.5%, 1.5%, and 3.0% in a non-irritating, dye-free, perfume-free, and fragrance-free vanishing cream vehicle. Ten mice used vehicle only and 10 were untreated. Each concentration of AC-11 and was applied daily to the backs of the mice prior to exposure to a 1,600-watt solar simulator used in this work (Solar Light Co. Philadelphia, PA) emitting (mainly Ultraviolet A (UVA) and B (UVB) radiation) duration of the experimental period with UVB wavelengths was filtered out with a 1.0 cm Schott WG 345 filter. AC-11 with a peak absorption at 200nm does act as a sun block. We tested for and focused on clinical appearance of mice and histological appearance of tumors in mice rather than metrics of radiation generated inflammation. Tumor progression scores were assigned as follows: 4+ = extensive tumor development; 3+ = early malignancies (raised palpable plaques)(early squamous cell cancers) 2+ = firm scaling, palpable keratosis (actinic keratoses); 1+ = light scaling with erythema. Following a total cumulative dose of 738 J/cm2, 85.7% all of the irradiated control animals, which did not receive AC-11 had precancerous actinic keratosis (AK)-type lesions (2+) (64.3% versus 42.9%) or early squamous cell carcinoma (SCC) (3+) (21.4% vs. 4.8%), in comparison with 47.7 % of AC-11-treated animals. There were no significant differences between the AC-11 groups. Three months after cessation of exposure to UVA radiation, the lesions in all but three of the 14 animals which were treated with AC-11 that were still evaluable irradiated with UVA radiation progressed to papillomas and frank

  3. Paclitaxel-loaded ethosomes®: potential treatment of squamous cell carcinoma, a malignant transformation of actinic keratoses.

    PubMed

    Paolino, Donatella; Celia, Christian; Trapasso, Elena; Cilurzo, Felisa; Fresta, Massimo

    2012-05-01

    Topical application of anticancer drugs for the treatment of malignancies represents a new challenge in dermatology, potentially being an alternative therapeutic approach for the efficacious treatment of non-melanoma skin cancer, that is, actinic keratoses, and malignant lesions of the skin caused by ultraviolet radiation. Anti-proliferative and antimitotic drugs, including many of the taxanes, are currently under investigation for the treatment of cutaneous malignant transformation of actinic keratoses, particularly the squamous cell carcinoma. Paclitaxel-loaded ethosomes® are proposed as topical drug delivery systems for the treatment of this pathology due to their suitable physicochemical characteristics and enhanced skin penetration ability for deep dermal delivery. Our in vitro data show that the skin application of paclitaxel-loaded ethosomes® improved the permeation of paclitaxel in a stratum corneum-epidermis membrane model and increased its anti-proliferative activity in a squamous cell carcinoma model as compared to the free drug. The results obtained encouraged the use of the paclitaxel-loaded ethosomes® as the formulation for the potential treatment of squamous cell carcinoma, a malignant transformation of actinic keratoses.

  4. Paclitaxel-loaded ethosomes®: potential treatment of squamous cell carcinoma, a malignant transformation of actinic keratoses.

    PubMed

    Paolino, Donatella; Celia, Christian; Trapasso, Elena; Cilurzo, Felisa; Fresta, Massimo

    2012-05-01

    Topical application of anticancer drugs for the treatment of malignancies represents a new challenge in dermatology, potentially being an alternative therapeutic approach for the efficacious treatment of non-melanoma skin cancer, that is, actinic keratoses, and malignant lesions of the skin caused by ultraviolet radiation. Anti-proliferative and antimitotic drugs, including many of the taxanes, are currently under investigation for the treatment of cutaneous malignant transformation of actinic keratoses, particularly the squamous cell carcinoma. Paclitaxel-loaded ethosomes® are proposed as topical drug delivery systems for the treatment of this pathology due to their suitable physicochemical characteristics and enhanced skin penetration ability for deep dermal delivery. Our in vitro data show that the skin application of paclitaxel-loaded ethosomes® improved the permeation of paclitaxel in a stratum corneum-epidermis membrane model and increased its anti-proliferative activity in a squamous cell carcinoma model as compared to the free drug. The results obtained encouraged the use of the paclitaxel-loaded ethosomes® as the formulation for the potential treatment of squamous cell carcinoma, a malignant transformation of actinic keratoses. PMID:22414731

  5. Evidence for field cancerisation treatment of actinic keratoses with topical diclofenac in hyaluronic acid.

    PubMed

    Ulrich, Martina; Pellacani, Giovanni; Ferrandiz, Carlos; Lear, John T

    2014-01-01

    Actinic keratosis (AK) is a common skin disease seen in daily practice. It is associated with a risk of progression to invasive squamous cell carcinoma and can be regarded as a marker of increased risk for non-melanoma skin cancer. The use of a field-directed treatment approach reflects the need to initiate early treatment over an affected area to prevent tumour development and local recurrence. Candidate field-directed treatments require a mechanism of action compatible with an effect on field cancerisation, immediate and long-term efficacy against visible lesions and efficacy against subclinical AK. Applicability to large treatment areas, tolerability compatible with long-term use, utility in organ transplant patients and, ideally, evidence of extended long-term activity may also be desirable. We review the evidence of a role for topical diclofenac sodium 3% administered in a 2.5% hyaluronic acid gel (diclofenac/HA) as field-directed treatment. Diclofenac/HA directly targets AK pathophysiology through multiple mechanisms, including induction of apoptosis, inhibition of angiogenesis and reduced inflammation. Clearance of visible field cancerisation is safely and rapidly achieved with a 90-day treatment course in patients with affected areas of up to 50 cm(2) and is associated with a ≥75% reduction in target lesion number score in 85% and 91% of patients, respectively, at 30 days and 1 year post-treatment. Following treatment of AK in high-risk transplant patients, 45% remained free of lesions in the treatment area at 2 years post-treatment. We conclude that diclofenac/HA fulfils most criteria necessary to be considered an appropriate candidate for a field-directed treatment in AK.

  6. Actinic Keratosis

    MedlinePlus

    ... rashes clinical tools newsletter | contact Share | Actinic Keratosis (Solar Keratosis) Information for adults A A A Actinic ... the touch. Overview Actinic keratoses, also known as solar keratoses, are small rough or scaly areas of ...

  7. Which plant for which skin disease? Part 2: Dermatophytes, chronic venous insufficiency, photoprotection, actinic keratoses, vitiligo, hair loss, cosmetic indications.

    PubMed

    Reuter, Juliane; Wölfle, Ute; Korting, Hans Christian; Schempp, Christoph

    2010-11-01

    This paper continues our review of scientifically evaluated plant extracts in dermatology. After plants effective against dermatophytes, botanicals with anti-edema effects in chronic venous insufficiency are discussed. There is good evidence from randomized clinical studies that plant extracts from grape vine leaves (Vitis vinifera), horse chestnut (Aesculus hippocastanum), sea pine (Pinus maritima) and butcher's broom (Ruscus aculeatus) can reduce edema in chronic venous insufficiency. Plant extracts from witch hazel (Hamamelis virginiana), green tea (Camellia sinensis), the fern Polypodium leucotomos and others contain antioxidant polyphenolic compounds that may protect the skin from sunburn and photoaging when administered topically or systemically. Extracts from the garden spurge (Euphorbia peplus) and from birch bark (Betula alba) have been shown to be effective in the treatment of actinic keratoses in phase II studies. Some plant extracts have also been investigated in the treatment of vitiligo, various forms of hair loss and pigmentation disorders, and in aesthetic dermatology. PMID:20707877

  8. Tumor-Preferential Induction of Immune Responses and Epidermal Cell Death in Actinic Keratoses by Ingenol Mebutate

    PubMed Central

    Zibert, John R.; Schön, Margarete; Hald, Andreas; Hansen, Maria H.; Litman, Thomas; Schön, Michael P.

    2016-01-01

    The rapid and strong clinical efficacy of the first-in-class, ingenol mebutate, against actinic keratosis (AK) has resulted in its recent approval. We conducted the first comprehensive analysis of the cellular and molecular mode of action of topical ingenol mebutate 0.05% gel in both AK and uninvolved skin of 26 patients in a phase I, single-center, open-label, within-patient comparison. As early as 1 day after application, ingenol mebutate induced profound epidermal cell death, along with a strong infiltrate of CD4+ and CD8+ T-cells, neutrophils, and macrophages. Endothelial ICAM-1 activation became evident after 2 days. The reaction pattern was significantly more pronounced in AK compared with uninvolved skin, suggesting a tumor-preferential mode of action. Extensive molecular analyses and transcriptomic profiling of mRNAs and microRNAs demonstrated alterations in gene clusters functionally associated with epidermal development, inflammation, innate immunity, and response to wounding. Ingenol mebutate reveals a unique mode of action linking directly to anti-tumoral effects. Trial Registration: ClinicalTrials.gov NCT01387711 PMID:27612149

  9. Actikerall™ (5-Fluorouracil 0.5% and Salicylic Acid 10%) Topical Solution for Patient-directed Treatment of Actinic Keratoses.

    PubMed

    Nguyen, H P; Rivers, J K

    2016-05-01

    Actinic keratosis (AK), a common cutaneous lesion with the potential to transform into squamous cell carcinoma, has traditionally been treated with ablative and/or surgical procedures. Recently, a topical formulation combining 0.5% 5-fluorouracil with 10% salicylic acid (5-FU-SA) was introduced in Europe under the trade name Actikerall™ for the treatment of grade I/II AKs. In a single randomized phase III trial, 5-FU-SA was shown to be superior to diclofenac 3% gel in hyaluronic acid, as measured by the histological clearance of one defined lesion (72% vs. 59.1%) and by complete clinical clearance (55.4% vs. 32.0%). 5-FU-SA should be applied once daily to a total area of up to 25 cm(2), which may include the lesion(s) and a small area of surrounding skin (rim of healthy skin should not exceed 0.5 cm), for up to 12 weeks. The most common side effects are local inflammation and pruritus at the application site, and no serious adverse effects have been reported to date. Now commercially available in Canada, 5-FU-SA represents a patientapplied therapeutic option for the treatment of both overt and subclinical AKs.

  10. Prevalence and awareness of actinic keratosis: barriers and opportunities.

    PubMed

    Rosen, Theodore; Lebwohl, Mark G

    2013-01-01

    Actinic keratoses (AKs) are common skin lesions that appear after long-term exposure to ultraviolet radiation. The presence of AKs is associated with an increased risk for development of nonmelanoma skin cancer. AKs vary widely in clinical and histologic presentation, which contributes to inadequate identification and presents challenges for consensus classification. Clinically adequate reduction in AK prevalence requires a multifaceted approach. There is a reasonable need to increase awareness and knowledge about AK, including symptoms, prevention, and associated risk of nonmelanoma skin cancer, especially among the public at large. Safe and effective treatment strategies are needed to optimize clearance of AKs, ideally to prevent progression to invasive cutaneous neoplasia. PMID:23228302

  11. A novel alpha kinase EhAK1 phosphorylates actin and regulates phagocytosis in Entamoeba histolytica.

    PubMed

    Mansuri, M Shahid; Bhattacharya, Sudha; Bhattacharya, Alok

    2014-10-01

    Phagocytosis plays a key role in nutrient uptake and virulence of the protist parasite Entamoeba histolytica. Phagosomes have been characterized by proteomics, and their maturation in the cells has been studied. However, there is so far not much understanding about initiation of phagocytosis and formation of phagosomes at the molecular level. Our group has been studying initiation of phagocytosis and formation of phagosomes in E. histolytica, and have described some of the molecules that play key roles in the process. Here we show the involvement of EhAK1, an alpha kinase and a SH3 domain containing protein in the pathway that leads to formation of phagosomes using red blood cell as ligand particle. A number of approaches, such as proteomics, biochemical, confocal imaging using specific antibodies or GFP tagged molecules, expression down regulation by antisense RNA, over expression of wild type and mutant proteins, were used to understand the role of EhAK1 in phagocytosis. EhAK1 was found in the phagocytic cups during the progression of cups, until closure of phagosomes, but not in the phagosomes themselves. It is recruited to the phagosomes through interaction with the calcium binding protein EhCaBP1. A reduction in phagocytosis was observed when EhAK1 was down regulated by antisense RNA, or by over expression of the kinase dead mutant. G-actin was identified as one of the major substrates of EhAK1. Phosphorylated actin preferentially accumulated at the phagocytic cups and over expression of a phosphorylation defective actin led to defects in phagocytosis. In conclusion, we describe an important component of the pathway that is initiated on attachment of red blood cells to E. histolytica cells. The main function of EhAK1 is to couple signalling events initiated after accumulation of EhC2PK to actin dynamics.

  12. A Novel Alpha Kinase EhAK1 Phosphorylates Actin and Regulates Phagocytosis in Entamoeba histolytica

    PubMed Central

    Mansuri, M. Shahid; Bhattacharya, Sudha; Bhattacharya, Alok

    2014-01-01

    Phagocytosis plays a key role in nutrient uptake and virulence of the protist parasite Entamoeba histolytica. Phagosomes have been characterized by proteomics, and their maturation in the cells has been studied. However, there is so far not much understanding about initiation of phagocytosis and formation of phagosomes at the molecular level. Our group has been studying initiation of phagocytosis and formation of phagosomes in E. histolytica, and have described some of the molecules that play key roles in the process. Here we show the involvement of EhAK1, an alpha kinase and a SH3 domain containing protein in the pathway that leads to formation of phagosomes using red blood cell as ligand particle. A number of approaches, such as proteomics, biochemical, confocal imaging using specific antibodies or GFP tagged molecules, expression down regulation by antisense RNA, over expression of wild type and mutant proteins, were used to understand the role of EhAK1 in phagocytosis. EhAK1 was found in the phagocytic cups during the progression of cups, until closure of phagosomes, but not in the phagosomes themselves. It is recruited to the phagosomes through interaction with the calcium binding protein EhCaBP1. A reduction in phagocytosis was observed when EhAK1 was down regulated by antisense RNA, or by over expression of the kinase dead mutant. G-actin was identified as one of the major substrates of EhAK1. Phosphorylated actin preferentially accumulated at the phagocytic cups and over expression of a phosphorylation defective actin led to defects in phagocytosis. In conclusion, we describe an important component of the pathway that is initiated on attachment of red blood cells to E. histolytica cells. The main function of EhAK1 is to couple signalling events initiated after accumulation of EhC2PK to actin dynamics. PMID:25299184

  13. The continued use of sunscreen prevents the development of actinic keratosis in aged Japanese subjects.

    PubMed

    Kunimoto, Kayo; Furukawa, Fukumi; Uede, Mikiko; Mizuno, Makoto; Yamamoto, Yuki

    2016-08-01

    It is well known that the trigger for actinic keratosis (AK) mainly depends on UV exposure. We evaluated the effects of long-term use of sunscreen on the histopathological and dermoscopic changes of AK in aged patients. Eighteen months use of sunscreen produced no change in the number of actinic keratoses or the advancement of histological grade. Although a significant decrease was not observed in the number of positive cells of p53, Ki-67 and COX-2 of the subjects who used sunscreen for 18 months, the downward tendencies of these proteins were observed. The continued use of sunscreen decreased the number of CD31-positive vessels significantly using the Chalkley method, and a significant improvement in scaling and vessel dots was found by dermoscopic study. Moreover, a relationship was found in the amount of sunscreen use and the number of actinic keratoses. Considering these results, it was thought that application of sunscreen reduces the risk of advancement of AK to higher grade AK and squamous cell carcinoma. PMID:27539900

  14. [Multiple bowenoid arsenic keratoses].

    PubMed

    Leyh, F; Rothlaender, J P

    1985-01-01

    Case report of multiple keratoses and chronic lymphatic leukemia after arsenic poisoning 30 years ago during a one-year exposure to copper acetoarsenate in a pesticide factory. Absorption through the skin with local arsenic skin damage is discussed. Etretinate therapy (1 mg/kg b. w.) was ineffective.

  15. Analysis of promoter hypermethylation of death-associated protein kinase and p16 tumor suppressor genes in actinic keratoses and squamous cell carcinomas of the skin.

    PubMed

    Tyler, Lisa N; Ai, Lingbao; Zuo, Chunlai; Fan, Chun-Yang; Smoller, Bruce R

    2003-07-01

    Death-associated protein kinase is a serine/threonine protein kinase implicated in promoting apoptosis and tumor suppression, whereas p16 is a tumor suppressor gene that inhibits cyclin-dependent kinase 4 and 6 activity and arrests the cell cycle in the G1 phase. Hypermethylation of death-associated protein kinase or p16 gene with resultant gene inactivation has been described in a wide variety of human cancers. Promoter methylation of the death-associated protein kinase and p16 gene has been found in about 55% and 30% cases of head and neck squamous cell carcinoma respectively but has not yet been analyzed in cutaneous premalignant and malignant lesions. A total of 33 cases were examined for evidence of death-associated protein kinase and p16 hypermethylation and these consist of 9 cases of spongiotic dermatitis as nonneoplastic skin control, 9 cases of actinic keratosis, 8 cases of squamous cell carcinoma in situ, and 7 cases of invasive squamous cell carcinoma. Death-associated protein kinase promoter methylation was detected in 1 case of squamous cell carcinoma in situ and 1 case of nonneoplastic skin control but none of the cases of invasive squamous cell carcinoma or actinic keratosis. P16 promoter methylation was detected in 1 case of invasive squamous cell carcinoma and 1 case of nonneoplastic skin control but none of the cases of squamous cell carcinoma in situ or actinic keratosis. Promoter hypermethylation of the death-associated protein kinase and p16 genes does not appear to play an important role in the development of cutaneous squamous cell carcinoma. The data thus suggest that the mechanisms of ultraviolet-induced cutaneous carcinomas differ from those involved in the development of head and neck squamous cell carcinoma, a malignant disease induced by tobacco and alcohol exposure.

  16. Update on photodynamic treatment for actinic keratosis.

    PubMed

    Wiegell, Stine Regin

    2015-01-01

    Photodynamic therapy (PDT) is an attractive treatment option for actinic keratoses (AKs), as large skin areas can be treated with high response rates and superior cosmetic outcome. The efficacy of 5-aminolevulinic acid (ALA)-PDT and methyl aminolevulinate (MAL)-PDT for AK has been proven in multiple studies, and this treatment is recommended in numerous consensus works and therapy guidelines. Moreover, a self-adhesive ALA patch has been approved for the PDT of AK. In a phase III study, ALA-patch-PDT was superior to cryotherapy and placebo in the treatment of mild to moderate AK on the face and scalp, and pre-treatment of the lesions and additional light occlusion was unnecessary when using the patch. ALA with a proprietary nanoemulsion is another newly marketed ALA gel that has been approved for the treatment of mild to moderate AK on the head. ALA was combined with a nanoemulsion to achieve increased chemical stability of the active ingredient and to enhance skin penetration. One study found that ALA was superior to MAL in the treatment of AK on the face or scalp. Daylight-PDT is a simpler and more tolerable treatment procedure for PDT, and three randomised studies have shown that daylight-PDT is an effective and pain-free treatment for AK; however, the procedure is limited by the need for a sufficient light dose and outdoor temperature. Ablative fractional laser resurfacing prior to MAL has been used to improve the PDT response of thick AK. However, more intense acute skin reactions and long-term adverse events in ablative fractional laser resurfacing-PDT compared with PDT-treated skin were found, which might limit the use of the intensified treatment.

  17. [Actinic Keratosis].

    PubMed

    Dejaco, D; Hauser, U; Zelger, B; Riechelmann, H

    2015-07-01

    Actinic keratosis is a cutaneous lesion characterized by proliferation of atypical epidermal keratinocytes due to prolonged exposure to exogenous factors such as ultraviolet radiation. AKs are in-situ-squamous cell carcinomas (PEC) of the skin. AK typically presents as erythematous, scaly patch or papule (classic AK), occasionally as thick, adherent scale on an erythematous base. Mostly fair-skinned adults are affected. AKs typically occur in areas of frequent sun exposure (balding scalp, face, "H-region", lateral neck, décolleté, dorsum of the hand and lower extremities). Actinic Cheilitis is the term used for AKs appearing on the lips. The diagnosis of AK is based on clinical examination including inspection and palpation. The typical palpable rough surface of AK often precedes a visible lesion. Dermoscopy may provide additional information. If diagnosis is uncertain and invasion suspected, biopsy and histopathologic evaluation should be performed. The potential for progression to invasive PECs mandates therapeutic intervention. Treatment options include topical and systemic therapies. Topical therapies are classified into physical, medical and combined physical-chemical approaches and a sequential combination of treatment modalities is possible. Topical-physical cryotherapy is the treatment of choice for isolated, non-hypertrophic AK. Topical-medical treatment, e. g. 5-fluoruracil (5FU) cream or Imiquomod or Ingenolmebutat application is used for multiple, non-hypertrophic AKs. For hypertrophic AKs, a dehorning pretreatment with salicinated vaseline is recommended. Isolated hypertrophic AKs often need cryotherapy with prolonged freezing time or several consecutive applications. Sequentially combined approaches are recommended for multiple, hypertrophic AKs. Photodynamic therapy (PDT) as example for a combined physical-chemical approach is an established treatment for multiple, non-hypertrophic and hypertrophic AKs. Prevention includes avoidance of sun and

  18. Efficacy of cryosurgery and 5-fluorouracil cream 0.5% combination therapy for the treatment of actinic keratosis.

    PubMed

    Hoover, William D; Jorizzo, Joseph L; Clark, Adele R; Feldman, Steven R; Holbrook, Judy; Huang, Karen E

    2014-11-01

    Actinic keratoses (AKs) are on a continuum of progression to squamous cell carcinoma (SCC). The most common AK treatment modalities are lesion-directed cryosurgery and field-directed therapy with 5-fluorouracil (5-FU); however, side effects can affect patient compliance. This study was performed to determine the efficacy and perceived side effects of combination treatment with cryosurgery and a shortened course of 5-FU cream 0.5% for AK lesions. Sixty participants with AK lesions underwent cryosurgery and were then randomized to apply 5-FU cream 0.5% or comparator cream once daily to the study area for 1 week. Participants were evaluated at weeks 3, 4, 8, and 26. After 8 weeks, treatment with cryosurgery and 5-FU cream 0.5% was more likely to result in complete clearance versus cryosurgery alone; however, no statistical difference was found in the complete clearance of AK lesions in the treatment group compared to cryosurgery alone at 26 weeks, while side effects in the treatment group were decreased. This study demonstrated the benefit of combination treatment of cryosurgery with 1 week of 5-FU compared to cryosurgery alone in clearing AK lesions for 2 months. This study shows promise for future studies with larger sample sizes to illustrate increased efficacy and decreased side effects with combination treatment of AKs with cryosurgery and 5-FU. PMID:25474455

  19. Mechanical and biological properties of keratose biomaterials.

    PubMed

    de Guzman, Roche C; Merrill, Michelle R; Richter, Jillian R; Hamzi, Rawad I; Greengauz-Roberts, Olga K; Van Dyke, Mark E

    2011-11-01

    The oxidized form of extractable human hair keratin proteins, commonly referred to as keratose, is gaining interest as a biomaterial for multiple tissue engineering studies including those directed toward peripheral nerve, spinal cord, skin, and bone regeneration. Unlike its disulfide cross-linked counterpart, kerateine, keratose does not possess a covalently cross-linked network structure and consequently displays substantially different characteristics. In order to understand its mode(s) of action and potential for clinical translatability, detailed characterization of the composition, physical properties, and biological responses of keratose biomaterials are needed. Keratose was obtained from end-cut human hair fibers by peracetic acid treatment, followed by base extraction, and subsequent dialysis. Analysis of lyophilized keratose powder determined that it contains 99% proteins by mass with amino acid content similar to human hair cortex. Metallic elements were also found in minute quantities. Protein oxidation led to disulfide bond cleavage and drastic reduction of free thiols due to conversion of sulfhydryl to sulfonic acid, chain fragmentation, and amino acid modifications. Mass spectrometry identified the major protein constituents as a heterogeneous mixture of 15 hair keratins (type I: K31-35 and K37-39, and type II: K81-86) with small amounts of epithelial keratins which exist in monomeric, dimeric, multimeric, and even degraded forms. Re-hydration with PBS enabled molecular assembly into an elastic solid-like hydrogel. Highly-porous scaffolds formed by lyophilization of the gel had the compression behavior of a cellular foam material and reverted back to gel upon wetting. Cytotoxicity assays showed that the EC50 for various cell lines were attained at 8-10 mg/mL keratose, indicating the non-toxic nature of the material. Implantation in mouse subcutaneous tissue pockets demonstrated that keratose resorption follows a rectangular hyperbolic regression

  20. Arsenic-induced keratoses and Bowen's disease.

    PubMed

    Watson, K; Creamer, D

    2004-01-01

    We report a patient with arsenic-induced keratoses and Bowen's disease. Chronic arsenicism may lead to both cutaneous and systemic neoplasms and patients should undergo regular long-term examination. Our patient had widespread cutaneous disease, which responded well to acitretin.

  1. Evaluation of ammonium lactate in the treatment of seborrheic keratoses.

    PubMed

    Klaus, M V; Wehr, R F; Rogers, R S; Russell, T J; Krochmal, L

    1990-02-01

    A double-blind, paired comparison study was used to evaluate treatment effects of 12% ammonium lactate lotion (Lac-Hydrin) against its vehicle on seborrheic keratoses. Fifty-eight volunteer patients, 37 to 82 years of age, were studied for 16 weeks. The patients had a minimum of two seborrheic keratoses at least 10 cm apart. They applied the medication twice daily. The lesions were evaluated for height, surface characteristics, color, and length with the use of 7X calibrated loupe, a template, skin replicas, and scanning electron microscopy. Lac-Hydrin 12% lotion significantly reduced the height (elevation) of seborrheic keratoses, and two seborrheic keratoses cleared completely; however, there was no statistically significant difference in the length, color, and surface characteristics between the study group and the control group. Skin replicas and scanning electron microscopy can be used to evaluate lesion surface characteristics, dimensions, and therapeutic effects.

  2. Comparative Study of Photodynamic Therapy with Topical Methyl Aminolevulinate versus 5-Aminolevulinic Acid for Facial Actinic Keratosis with Long-Term Follow-Up

    PubMed Central

    Ko, Dong-Yeob; Kim, Ki-Ho

    2014-01-01

    Background Few studies have compared the efficacy, cosmetic outcomes, and adverse events between 5-aminolevulinic acid photodynamic therapy (ALA-PDT) and methyl aminolevulinate-PDT (MAL-PDT) for actinic keratoses (AKs) in Asian ethnic populations with dark-skin. Objective We retrospectively compared the long-term efficacy, recurrence rates, cosmetic outcomes, and safety of ALA-PDT versus MAL-PDT for facial AKs in Koreans. Methods A total of 222 facial AKs in 58 patients were included in this study. A total of 153 lesions (29 patients) were treated with 5-ALA, and 69 lesions (29 patients) with MAL. ALA and MAL creams were applied for 6 hours and 3 hours, respectively; the lesions were then illuminated with a halogen lamp at 150 J/cm2 for ALA-PDT and a diode lamp at 37 J/cm2 for MAL-PDT. Results The complete response rates of ALA-PDT and MAL-PDT were 56.9% and 50.7%, respectively, with no significant difference at 12 months after treatment. No significant difference in recurrence rates was observed between the 2 PDT modalities at either 6 or 12 months after treatment. There was no significant difference in the cosmetic outcomes between the 2 treatment modalities at 12 months after PDT. However, ALA-PDT caused significantly more painful than MAL-PDT (p=0.005). The adverse events were mild to moderate, transient, and self-limiting for both modalities. Conclusion MAL-PDT was similar to ALA-PDT in terms of long-term efficacy, recurrence rates, cosmetic outcomes, and adverse events; however, it was a significantly less painful procedure than ALA-PDT in our study. PMID:24966631

  3. Oral nicotinamide and actinic keratosis: a supplement success story.

    PubMed

    Kim, Burcu; Halliday, Gary M; Damian, Diona L

    2015-01-01

    Nicotinamide has shown potential as a safe and effective intervention for the prevention of malignant and premalignant skin lesions. Recent studies have shown that nicotinamide, in both oral and topical forms, is able to prevent ultraviolet-induced immunosuppression in humans [1,2,3] and mice [4,5]. Immunosuppression is a known factor for the progression of premalignant lesions, such as actinic keratosis [6]. Murine studies have shown that nicotinamide is also able to protect against photocarcinogenesis [4,5]. Preliminary human studies suggest that nicotinamide may help prevent skin cancers and enhance the regression of actinic keratoses.

  4. Oral nicotinamide and actinic keratosis: a supplement success story.

    PubMed

    Kim, Burcu; Halliday, Gary M; Damian, Diona L

    2015-01-01

    Nicotinamide has shown potential as a safe and effective intervention for the prevention of malignant and premalignant skin lesions. Recent studies have shown that nicotinamide, in both oral and topical forms, is able to prevent ultraviolet-induced immunosuppression in humans [1,2,3] and mice [4,5]. Immunosuppression is a known factor for the progression of premalignant lesions, such as actinic keratosis [6]. Murine studies have shown that nicotinamide is also able to protect against photocarcinogenesis [4,5]. Preliminary human studies suggest that nicotinamide may help prevent skin cancers and enhance the regression of actinic keratoses. PMID:25561219

  5. Defining Field Cancerization of the Skin Using Noninvasive Optical Coherence Tomography Imaging to Detect and Monitor Actinic Keratosis in Ingenol Mebutate 0.015%- Treated Patients

    PubMed Central

    Schwartz, Michelle; Feldman, Eleanor; Bieber, Amy; Bienenfeld, Amanda; Nandanan, Naveen; Siegel, Daniel M.

    2016-01-01

    Objective: The objective of this study was to assess the ability of optical coherence tomography to detect clinical and subclinical actinic keratoses confirmed by histopathology. The efficacy of ingenol mebutate treatment of actinic keratosis was also evaluated using optical coherence tomography, and correlation of treatment efficacy with severity of local skin reactions was determined. Design: Single-arm, open-label, split-face study. Setting: Hospital outpatient clinic. Participants: Male subjects (N=30) with seven actinic keratoses. Measurements: A suspected actinic keratosis and the normal-appearing, perilesional skin were imaged, biopsied for histopathologic analysis, and the results compared with the clinical and a blinded optical coherence tomography diagnosis. Treatment with ingenol mebutate gel 0.015% was randomly administered to three clinically suspected actinic keratoses and the perilesional skin; three additional, suspected actinic keratoses lesions and perilesional areas were left untreated. Clinical and optical coherence tomography images were obtained for all lesions. Severity of local skin reactions was recorded to evaluate the relationship between local skin reaction and treatment effect. Results: Optical coherence tomography analysis had a 100-percent (28/28) correlation with the clinical diagnosis of actinic keratosis and detected 16 of 22 (73%) histopathologically confirmed subclinical lesions from perilesional skin sites. By optical coherence tomography assessment, the clearance rate for clinically observed lesions was 76 percent for ingenol mebutate-treated areas versus 11 percent for untreated areas; the clearance rate for treated subclinical lesions was 88 percent versus 43 percent for untreated areas. Clearance rates did not vary with the severity of the local response. Conclusion: Optical coherence tomography is effective at detecting clinical and subclinical actinic keratoses and monitoring their response to treatment. PMID:27386042

  6. Actinic keratosis

    MedlinePlus

    Solar keratosis; Sun-induced skin changes - keratosis; Keratosis - actinic (solar) ... Actinic keratosis is caused by exposure to sunlight. You are more likely to develop it if you: Have fair skin, blue or green eyes, or blond or red hair Had a ...

  7. Actinic Cheilitis

    MedlinePlus

    ... is a precancerous condition related to cumulative lifetime sun exposure. The lower lip is most often affected. Individuals ... Wearing barrier clothing (eg, wide-brimmed hats) and sunscreen-containing lip balms can aid in preventing actinic ...

  8. Computational Study of the Binding Mechanism of Actin-Depolymerizing Factor 1 with Actin in Arabidopsis thaliana

    PubMed Central

    Wang, Xue; Dong, Chun-Hai; Yang, Jian Ming; Yao, Xiao Jun

    2016-01-01

    Actin is a highly conserved protein. It plays important roles in cellular function and exists either in the monomeric (G-actin) or polymeric form (F-actin). Members of the actin-depolymerizing factor (ADF)/cofilin protein family bind to both G-actin and F-actin and play vital roles in actin dynamics by manipulating the rates of filament polymerization and depolymerization. It has been reported that the S6D and R98A/K100A mutants of actin-depolymerizing factor 1 (ADF1) in Arabidopsis thaliana decreased the binding affinity of ADF for the actin monomer. To investigate the binding mechanism and dynamic behavior of the ADF1–actin complex, we constructed a homology model of the AtADF1–actin complex based on the crystal structure of AtADF1 and the twinfilin C-terminal ADF-H domain in a complex with a mouse actin monomer. The model was then refined for subsequent molecular dynamics simulations. Increased binding energy of the mutated system was observed using the Molecular Mechanics Generalized Born Surface Area and Poisson–Boltzmann Surface Area (MM-GB/PBSA) methods. To determine the residues that make decisive contributions to the ADF1 actin-binding affinity, per-residue decomposition and computational alanine scanning analyses were performed, which provided more detailed information on the binding mechanism. Root-mean-square fluctuation and principal component analyses confirmed that the S6D and R98A/K100A mutants induced an increased conformational flexibility. The comprehensive molecular insight gained from this study is of great importance for understanding the binding mechanism of ADF1 and G-actin. PMID:27414648

  9. Computational Study of the Binding Mechanism of Actin-Depolymerizing Factor 1 with Actin in Arabidopsis thaliana.

    PubMed

    Du, Juan; Wang, Xue; Dong, Chun-Hai; Yang, Jian Ming; Yao, Xiao Jun

    2016-01-01

    Actin is a highly conserved protein. It plays important roles in cellular function and exists either in the monomeric (G-actin) or polymeric form (F-actin). Members of the actin-depolymerizing factor (ADF)/cofilin protein family bind to both G-actin and F-actin and play vital roles in actin dynamics by manipulating the rates of filament polymerization and depolymerization. It has been reported that the S6D and R98A/K100A mutants of actin-depolymerizing factor 1 (ADF1) in Arabidopsis thaliana decreased the binding affinity of ADF for the actin monomer. To investigate the binding mechanism and dynamic behavior of the ADF1-actin complex, we constructed a homology model of the AtADF1-actin complex based on the crystal structure of AtADF1 and the twinfilin C-terminal ADF-H domain in a complex with a mouse actin monomer. The model was then refined for subsequent molecular dynamics simulations. Increased binding energy of the mutated system was observed using the Molecular Mechanics Generalized Born Surface Area and Poisson-Boltzmann Surface Area (MM-GB/PBSA) methods. To determine the residues that make decisive contributions to the ADF1 actin-binding affinity, per-residue decomposition and computational alanine scanning analyses were performed, which provided more detailed information on the binding mechanism. Root-mean-square fluctuation and principal component analyses confirmed that the S6D and R98A/K100A mutants induced an increased conformational flexibility. The comprehensive molecular insight gained from this study is of great importance for understanding the binding mechanism of ADF1 and G-actin. PMID:27414648

  10. First report of fossil "keratose" demosponges in Phanerozoic carbonates: preservation and 3-D reconstruction

    NASA Astrophysics Data System (ADS)

    Luo, Cui; Reitner, Joachim

    2014-06-01

    Fossil record of Phanerozoic non-spicular sponges, beside of being important with respect to the lineage evolution per se, could provide valuable references for the investigation of Precambrian ancestral animal fossils. However, although modern phylogenomic studies resolve non-spicular demosponges as the sister group of the remaining spiculate demosponges, the fossil record of the former is extremely sparse or unexplored compared to that of the latter; the Middle Cambrian Vauxiidae Walcott 1920, is the only confirmed fossil taxon of non-spicular demosponges. Here, we describe carbonate materials from Devonian (Upper Givetian to Lower Frasnian) bioherms of northern France and Triassic (Anisian) microbialites of Poland that most likely represent fossil remnants of keratose demosponges. These putative fossils of keratose demosponges are preserved as automicritic clumps. They are morphologically distinguishable from microbial fabrics but similar to other spiculate sponge fossils, except that the skeletal elements consist of fibrous networks instead of assembled spicules. Consistent with the immunological behavior of sponges, these fibrous skeletons often form a rim at the edge of the automicritic aggregate, separating the inner part of the aggregate from foreign objects. To confirm the architecture of these fibrous networks, two fossil specimens and a modern thorectid sponge for comparison were processed for three-dimensional (3-D) reconstruction using serial grinding tomography. The resulting fossil reconstructions are three-dimensionally anastomosing, like modern keratose demosponges, but their irregular and nonhierarchical meshes indicate a likely verongid affinity, although a precise taxonomic conclusion cannot be made based on the skeletal architecture alone. This study is a preliminary effort, but an important start to identify fossil non-spicular demosponges in carbonates and to re-evaluate their fossilization potential.

  11. First report of fossil "keratose" demosponges in Phanerozoic carbonates: preservation and 3-D reconstruction.

    PubMed

    Luo, Cui; Reitner, Joachim

    2014-06-01

    Fossil record of Phanerozoic non-spicular sponges, beside of being important with respect to the lineage evolution per se, could provide valuable references for the investigation of Precambrian ancestral animal fossils. However, although modern phylogenomic studies resolve non-spicular demosponges as the sister group of the remaining spiculate demosponges, the fossil record of the former is extremely sparse or unexplored compared to that of the latter; the Middle Cambrian Vauxiidae Walcott 1920, is the only confirmed fossil taxon of non-spicular demosponges. Here, we describe carbonate materials from Devonian (Upper Givetian to Lower Frasnian) bioherms of northern France and Triassic (Anisian) microbialites of Poland that most likely represent fossil remnants of keratose demosponges. These putative fossils of keratose demosponges are preserved as automicritic clumps. They are morphologically distinguishable from microbial fabrics but similar to other spiculate sponge fossils, except that the skeletal elements consist of fibrous networks instead of assembled spicules. Consistent with the immunological behavior of sponges, these fibrous skeletons often form a rim at the edge of the automicritic aggregate, separating the inner part of the aggregate from foreign objects. To confirm the architecture of these fibrous networks, two fossil specimens and a modern thorectid sponge for comparison were processed for three-dimensional (3-D) reconstruction using serial grinding tomography. The resulting fossil reconstructions are three-dimensionally anastomosing, like modern keratose demosponges, but their irregular and nonhierarchical meshes indicate a likely verongid affinity, although a precise taxonomic conclusion cannot be made based on the skeletal architecture alone. This study is a preliminary effort, but an important start to identify fossil non-spicular demosponges in carbonates and to re-evaluate their fossilization potential.

  12. First report of fossil "keratose" demosponges in Phanerozoic carbonates: preservation and 3-D reconstruction.

    PubMed

    Luo, Cui; Reitner, Joachim

    2014-06-01

    Fossil record of Phanerozoic non-spicular sponges, beside of being important with respect to the lineage evolution per se, could provide valuable references for the investigation of Precambrian ancestral animal fossils. However, although modern phylogenomic studies resolve non-spicular demosponges as the sister group of the remaining spiculate demosponges, the fossil record of the former is extremely sparse or unexplored compared to that of the latter; the Middle Cambrian Vauxiidae Walcott 1920, is the only confirmed fossil taxon of non-spicular demosponges. Here, we describe carbonate materials from Devonian (Upper Givetian to Lower Frasnian) bioherms of northern France and Triassic (Anisian) microbialites of Poland that most likely represent fossil remnants of keratose demosponges. These putative fossils of keratose demosponges are preserved as automicritic clumps. They are morphologically distinguishable from microbial fabrics but similar to other spiculate sponge fossils, except that the skeletal elements consist of fibrous networks instead of assembled spicules. Consistent with the immunological behavior of sponges, these fibrous skeletons often form a rim at the edge of the automicritic aggregate, separating the inner part of the aggregate from foreign objects. To confirm the architecture of these fibrous networks, two fossil specimens and a modern thorectid sponge for comparison were processed for three-dimensional (3-D) reconstruction using serial grinding tomography. The resulting fossil reconstructions are three-dimensionally anastomosing, like modern keratose demosponges, but their irregular and nonhierarchical meshes indicate a likely verongid affinity, although a precise taxonomic conclusion cannot be made based on the skeletal architecture alone. This study is a preliminary effort, but an important start to identify fossil non-spicular demosponges in carbonates and to re-evaluate their fossilization potential. PMID:24763744

  13. Behavioural dynamics of a clinical trial of sunscreens for reducing solar keratoses in Victoria, Australia.

    PubMed Central

    Cockburn, J; Thompson, S C; Marks, R; Jolley, D; Schofield, P; Hill, D

    1997-01-01

    OBJECTIVE: To determine whether the behaviour of participants based on perception of treatment group in a randomised trial contributed to clinical outcome. DESIGN: A double blind randomised controlled trial of the effect of daily application of SPF 17 broad spectrum sunscreen cream (or placebo) on solar keratoses. SETTING: A rural city in Victoria, Australia. Residents aged 40 years or over were invited by letter to attend for a skin cancer screening check. Of these, 588 people with between one and 30 solar keratoses enrolled in the trial and 431 completed the trial, which extended over a six month period that included summer. Participants' perceptions of their treatment allocation, adherence with the treatment regimen, adoption of other sun protection behaviours, side effects, and perceptions of change in condition were measured at two monthly intervals. RESULTS: There were no significant differences between those who completed the study and those that did not for sex, age, treatment group, skin type, number of solar keratoses or correct perception of treatment group. Thirty per cent of those completing the study correctly guessed their treatment allocation, and people were just as likely to be right as to be wrong when they stated their opinion about their treatment allocation (z = 1.04; p = 0.15). Study group, skin type, amount of time spent outdoors, presence of side effects, perceptions of change in skin condition did not significantly predict correct perception of treatment allocation. Multivariate analysis of variance indicated that adoption of other sun protection and adherence with cream use were not significantly affected by actual treatment allocation, correct perception of treatment allocation nor by their interaction. Poisson regression analysis showed a significantly lower difference ratio of solar keratoses in the sunscreen group compared with the placebo base cream group (OR 0.55; CI = 0.46, 0.64), and for women compared with men (OR = 0.76; CI = 0

  14. Thickness of Actinic Keratosis Does Not Predict Dysplasia Severity or P53 Expression

    PubMed Central

    Heerfordt, Ida M.; Nissen, Christoffer V.; Poulsen, Thomas; Philipsen, Peter A.; Wulf, Hans Christian

    2016-01-01

    The severity of dysplasia and expression of p53 in actinic keratosis (AK) is of importance for the transformation to squamous cell carcinoma. It is assumed that it is most important to treat thick AKs as they are believed to be more dysplastic than thin AKs. However, a relation between AK thickness and dysplasia or the expression of p53 has never been demonstrated. The aim of this study was to investigate this possible relation. Sixty-six AKs were included for clinical and histological examination. Prior to performing a punch biopsy, the clinical thickness of each AK was measured objectively using two scale bars with a thickness of 0.5 mm and 1 mm. Subsequently, the thickness of the epidermis, the severity of dysplasia and the expression of p53 were assessed histologically. We found a strong and significant positive correlation between measured clinical thickness of the AKs and the histological thickness of epidermis (p < 0.0001). However, the clinical thickness did not correlate with either the severity of dysplasia (p = 0.7) or the expression of p53 (p = 0.5). In conclusion, thin AKs show the same severity of dysplasia and expression of p53 as thicker AK lesions. Consequently, clinical thickness cannot predict aggressiveness. PMID:27670104

  15. Actin in Herpesvirus Infection

    PubMed Central

    Roberts, Kari L.; Baines, Joel D.

    2011-01-01

    Actin is important for a variety of cellular processes, including uptake of extracellular material and intracellular transport. Several emerging lines of evidence indicate that herpesviruses exploit actin and actin-associated myosin motors for viral entry, intranuclear transport of capsids, and virion egress. The goal of this review is to explore these processes and to highlight potential future directions for this area of research. PMID:21994736

  16. Actin Rings of Power.

    PubMed

    Schwayer, Cornelia; Sikora, Mateusz; Slováková, Jana; Kardos, Roland; Heisenberg, Carl-Philipp

    2016-06-20

    Circular or ring-like actin structures play important roles in various developmental and physiological processes. Commonly, these rings are composed of actin filaments and myosin motors (actomyosin) that, upon activation, trigger ring constriction. Actomyosin ring constriction, in turn, has been implicated in key cellular processes ranging from cytokinesis to wound closure. Non-constricting actin ring-like structures also form at cell-cell contacts, where they exert a stabilizing function. Here, we review recent studies on the formation and function of actin ring-like structures in various morphogenetic processes, shedding light on how those different rings have been adapted to fulfill their specific roles. PMID:27326928

  17. Molecular discrimination of cutaneous squamous cell carcinoma from actinic keratosis and normal skin.

    PubMed

    Ra, Seong Hui; Li, Xinmin; Binder, Scott

    2011-07-01

    Actinic keratosis is widely believed to be a neoplastic lesion and a precursor to invasive squamous cell carcinoma. However, there has been some debate as to whether actinic keratosis is in fact actually squamous cell carcinoma and should be treated as such. As the clinical management and prognosis of patients is widely held to be different for each of these lesions, our goal was to identify unique gene signatures using DNA microarrays to discriminate among normal skin, actinic keratosis, and squamous cell carcinoma, and examine the molecular pathways of carcinogenesis involved in the progression from normal skin to squamous cell carcinoma. Formalin-fixed and paraffin-embedded blocks of skin: five normal skins (pooled), six actinic keratoses, and six squamous cell carcinomas were retrieved. The RNA was extracted and amplified. The labeled targets were hybridized to the Affymetrix human U133plus2.0 array and the acquisition and initial quantification of array images were performed using the GCOS (Affymetrix). The subsequent data analyses were performed using DNA-Chip Analyzer and Partek Genomic Suite 6.4. Significant differential gene expression (>2 fold change, P<0.05) was seen with 382 differentially expressed genes between squamous cell carcinoma and normal skin, 423 differentially expressed genes between actinic keratosis and normal skin, and 9 differentially expressed genes between actinic keratosis and squamous cell carcinoma. The differentially expressed genes offer the possibility of using DNA microarrays as a molecular diagnostic tool to distinguish between normal skin, actinic keratosis, and squamous cell carcinoma. In addition, the differentially expressed genes and their molecular pathways could be potentially used as prognostic markers or targets for future therapeutic innovations. PMID:21743436

  18. Seborrheic Keratoses

    MedlinePlus

    ... 2006-2013 Logical Images, Inc. All rights reserved. Advertising Notice This Site and third parties who place ... would like to obtain more information about these advertising practices and to make choices about online behavioral ...

  19. Efficacy of a photolyase-based device in the treatment of cancerization field in patients with actinic keratosis and non-melanoma skin cancer.

    PubMed

    Puviani, M; Barcella, A; Milani, M

    2013-12-01

    Eryfotona AK-NMSC (ISDIN Spain) is a film-forming medical device in cream or fluid formulation containing the DNA-repair enzyme photolyase and high-protection UV filters in liposomes (repairsomes) indicated in the treatment of cancerization field in patients with actinic keratosis (AK) or non-melanoma skin cancer (NMSC). Photolyase is an enzyme that recognizes and directly repairs UV-induced DNA damage. The most common UV-induced DNA damage is the formation of cyclobutane pyrimidine dimers (CPD). Clinical studies evaluating the histological and cellular effects of Eryfotona AK-NMSC have shown a potential benefit in the treatment of the cancerization field in AK patients. In particular the use of Eryfotona AK-NMSC improves the confocal microscopic appearance of skin at the cancerization field level. In addition, Eryfotona AK-NMSC improves the p53 gene expression at keratinocyte level. In this study we reported a series of 6 cases of patients with AK or NMSC lesions treated with Eryfotona AK-NMSC fluid, both as coadjuvant and as single treatment, applied twice daily in the affected area with photograph documentation. Clinical photographs of the skin lesions at baseline and after Eryfotona AK-NMSC treatment were taken in all cases using a high-definition digital camera. Six patients with multiple AK lesions of the scalp or face with or without NMSC were treated for a mean of 1-3 months with Eryfotona AK-NMSC fluid formulation. Image documentations before and after treatment of this clinical series show a great improvement in AK lesions count and of cancerization field. This clinical series supports the clinical efficacy of the use of photolyase and high-protection UV filters in the treatment of cancerization field and AK lesions in patients with actinic damage. PMID:24442053

  20. Direct dynamin–actin interactions regulate the actin cytoskeleton

    PubMed Central

    Gu, Changkyu; Yaddanapudi, Suma; Weins, Astrid; Osborn, Teresia; Reiser, Jochen; Pollak, Martin; Hartwig, John; Sever, Sanja

    2010-01-01

    The large GTPase dynamin assembles into higher order structures that are thought to promote endocytosis. Dynamin also regulates the actin cytoskeleton through an unknown, GTPase-dependent mechanism. Here, we identify a highly conserved site in dynamin that binds directly to actin filaments and aligns them into bundles. Point mutations in the actin-binding domain cause aberrant membrane ruffling and defective actin stress fibre formation in cells. Short actin filaments promote dynamin assembly into higher order structures, which in turn efficiently release the actin-capping protein (CP) gelsolin from barbed actin ends in vitro, allowing for elongation of actin filaments. Together, our results support a model in which assembled dynamin, generated through interactions with short actin filaments, promotes actin polymerization via displacement of actin-CPs. PMID:20935625

  1. Dynamin at actin tails.

    PubMed

    Lee, Eunkyung; De Camilli, Pietro

    2002-01-01

    Dynamin, the product of the shibire gene of Drosophila, is a GTPase critically required for endocytosis. Some studies have suggested a functional link between dynamin and the actin cytoskeleton. This link is of special interest, because there is evidence implicating actin dynamics in endocytosis. Here we show that endogenous dynamin 2, as well as green fluorescence protein fusion proteins of both dynamin 1 and 2, is present in actin comets generated by Listeria or by type I PIP kinase (PIPK) overexpression. In PIPK-induced tails, dynamin is further enriched at the interface between the tails and the moving organelles. Dynamin mutants harboring mutations in the GTPase domain inhibited nucleation of actin tails induced by PIPK and moderately reduced their speed. Although dynamin localization to the tails required its proline-rich domain, expression of a dynamin mutant lacking this domain also diminished tail formation. In addition, this mutant disrupted a membrane-associated actin scaffold (podosome rosette) previously shown to include dynamin. These findings suggest that dynamin is part of a protein network that controls nucleation of actin from membranes. At endocytic sites, dynamin may couple the fission reaction to the polymerization of an actin pool that functions in the separation of the endocytic vesicles from the plasma membrane. PMID:11782545

  2. Actin Mechanics and Fragmentation*

    PubMed Central

    De La Cruz, Enrique M.; Gardel, Margaret L.

    2015-01-01

    Cell physiological processes require the regulation and coordination of both mechanical and dynamical properties of the actin cytoskeleton. Here we review recent advances in understanding the mechanical properties and stability of actin filaments and how these properties are manifested at larger (network) length scales. We discuss how forces can influence local biochemical interactions, resulting in the formation of mechanically sensitive dynamic steady states. Understanding the regulation of such force-activated chemistries and dynamic steady states reflects an important challenge for future work that will provide valuable insights as to how the actin cytoskeleton engenders mechanoresponsiveness of living cells. PMID:25957404

  3. Actin Automata with Memory

    NASA Astrophysics Data System (ADS)

    Alonso-Sanz, Ramón; Adamatzky, Andy

    Actin is a globular protein which forms long polar filaments in eukaryotic. The actin filaments play the roles of cytoskeleton, motility units, information processing and learning. We model actin filament as a double chain of finite state machines, nodes, which take states “0” and “1”. The states are abstractions of absence and presence of a subthreshold charge on actin units corresponding to the nodes. All nodes update their state in parallel to discrete time. A node updates its current state depending on states of two closest neighbors in the node chain and two closest neighbors in the complementary chain. Previous models of actin automata consider momentary state transitions of nodes. We enrich the actin automata model by assuming that states of nodes depend not only on the current states of neighboring node but also on their past states. Thus, we assess the effect of memory of past states on the dynamics of acting automata. We demonstrate in computational experiments that memory slows down propagation of perturbations, decrease entropy of space-time patterns generated, transforms traveling localizations to stationary oscillators, and stationary oscillations to still patterns.

  4. The Molecular Evolution of Actin

    PubMed Central

    Hightower, Robin C.; Meagher, Richard B.

    1986-01-01

    We have investigated the molecular evolution of plant and nonplant actin genes comparing nucleotide and amino acid sequences of 20 actin genes. Nucleotide changes resulting in amino acid substitutions (replacement substitutions) ranged from 3–7% for all pairwise comparisons of animal actin genes with the following exceptions. Comparisons between higher animal muscle actin gene sequences and comparisons between higher animal cytoplasmic actin gene sequences indicated <3% divergence. Comparisons between plant and nonplant actin genes revealed, with two exceptions, 11–15% replacement substitution. In the analysis of plant actins, replacement substitution between soybean actin genes SAc1, SAc3, SAc4 and maize actin gene MAc1 ranged from 8–10%, whereas these members within the soybean actin gene family ranged from 6–9% replacement substitution. The rate of sequence divergence of plant actin sequences appears to be similar to that observed for animal actins. Furthermore, these and other data suggest that the plant actin gene family is ancient and that the families of soybean and maize actin genes have diverged from a single common ancestral plant actin gene that originated long before the divergence of monocots and dicots. The soybean actin multigene family encodes at least three classes of actin. These classes each contain a pair of actin genes that have been designated kappa (SAc1, SAc6), lambda (SAc2, SAc4) and mu (SAc3, SAc7). The three classes of soybean actin are more divergent in nucleotide sequence from one another than higher animal cytoplasmic actin is divergent from muscle actin. The location and distribution of amino acid changes were compared between actin proteins from all sources. A comparison of the hydropathy of all actin sequences, except from Oxytricha, indicated a strong similarity in hydropathic character between all plant and nonplant actins despite the greater number of replacement substitutions in plant actins. These protein sequence

  5. Comparison of three light doses in the photodynamic treatment of actinic keratosis using mathematical modeling

    NASA Astrophysics Data System (ADS)

    Vignion-Dewalle, Anne-Sophie; Betrouni, Nacim; Tylcz, Jean-Baptiste; Vermandel, Maximilien; Mortier, Laurent; Mordon, Serge

    2015-05-01

    Photodynamic therapy (PDT) is an emerging treatment modality for various diseases, especially for cancer therapy. Although high efficacy is demonstrated for PDT using standardized protocols in nonhyperkeratotic actinic keratoses, alternative light doses expected to increase efficiency, to reduce adverse effects or to expand the use of PDT, are still being evaluated and refined. We propose a comparison of the three most common light doses in the treatment of actinic keratosis with 5-aminolevulinic acid PDT through mathematical modeling. The proposed model is based on an iterative procedure that involves determination of the local fluence rate, updating of the local optical properties, and estimation of the local damage induced by the therapy. This model was applied on a simplified skin sample model including an actinic keratosis lesion, with three different light doses (red light dose, 37 J/cm2, 75 mW/cm2, 500 s blue light dose, 10 J/cm2, 10 mW/cm2, 1000 s and daylight dose, 9000 s). Results analysis shows that the three studied light doses, although all efficient, lead to variable local damage. Defining reference damage enables the nonoptimal parameters for the current light doses to be refined and the treatment to be more suitable.

  6. Comparison of three light doses in the photodynamic treatment of actinic keratosis using mathematical modeling.

    PubMed

    Vignion-Dewalle, Anne-Sophie; Betrouni, Nacim; Tylcz, Jean-Baptiste; Vermandel, Maximilien; Mortier, Laurent; Mordon, Serge

    2015-05-01

    Photodynamic therapy (PDT) is an emerging treatment modality for various diseases, especially for cancer therapy. Although high efficacy is demonstrated for PDT using standardized protocols in nonhyperkeratotic actinic keratoses, alternative light doses expected to increase efficiency, to reduce adverse effects or to expand the use of PDT, are still being evaluated and refined. We propose a comparison of the three most common light doses in the treatment of actinic keratosis with 5-aminolevulinic acid PDT through mathematical modeling. The proposed model is based on an iterative procedure that involves determination of the local fluence rate, updating of the local optical properties, and estimation of the local damage induced by the therapy. This model was applied on a simplified skin sample model including an actinic keratosis lesion, with three different light doses (red light dose, 37 J∕cm2, 75 mW∕cm2, 500 s; blue light dose, 10 J∕cm2, 10 mW∕cm2, 1000 s; and daylight dose, 9000 s). Results analysis shows that the three studied light doses, although all efficient, lead to variable local damage. Defining reference damage enables the nonoptimal parameters for the current light doses to be refined and the treatment to be more suitable.

  7. New therapeutic options for actinic keratosis and basal cell carcinoma.

    PubMed

    Sligh, James E

    2014-06-01

    Actinic keratosis (AK) is a common premalignant skin lesion that is frequently treated by cryosurgery. Basal cell carcinoma is the most common malignancy of man, and early-stage lesions are usually cured via surgery. Advanced basal cell carcinoma may require more extensive surgery resulting in deformity, and many advanced lesions cannot be treated surgically. Several recent developments have improved therapeutic options for both conditions. Cryosurgery is still a mainstay of treatment for AK, but the introduction of effective topical agents, imiquimod cream and ingenol mebutate, has provided alternatives to cryosurgery. For advanced basal cell carcinoma, the small-molecule inhibitor vismodegib has proven to be an effective therapy for lesions that are not amenable to surgery and has demonstrated ability to achieve dramatic improvement in advanced, potentially disfiguring cancer. PMID:25268601

  8. Combining morphometrics with molecular taxonomy: how different are similar foliose keratose sponges from the Australian tropics?

    PubMed

    Abdul Wahab, M A; Fromont, J; Whalan, S; Webster, N; Andreakis, N

    2014-04-01

    Sponge taxonomy can be challenging as many groups exhibit extreme morphological plasticity induced by local environmental conditions. Foliose keratose sponges of the sub-family Phyllospongiinae (Dictyoceratida, Thorectidae: Strepsichordaia, Phyllospongia and Carteriospongia) are commonly found in intertidal and subtidal habitats of the Indo-Pacific. Lacking spicules, these sponges can be difficult to differentiate due to the lack of reliable morphological characters for species delineation. We use molecular phylogenies inferred from the nuclear Internal Transcribed Spacer 2 region (ITS2) and morphometrics (19 characters; 52 character states) to identify evolutionarily significant units (ESUs; sensu Moritz) within foliose Phyllosponginiids collected from seven geographic locations across tropical eastern and Western Australia. The ITS2 topology was congruent with the tree derived from Bayesian inference of discrete morphological characters supporting expected taxonomic relationships at the genus level and the identification of five ESUs. However, phylogenies inferred from the ITS2 marker revealed multiple sequence clusters, some of which were characterised by distinct morphological features and specific geographic ranges. Our results are discussed in light of taxonomic incongruences within this study, hidden sponge diversity and the role of vicariant events in influencing present day distribution patterns.

  9. Apoptosis in seborrheic keratoses: an open door to a new dermoscopic score.

    PubMed

    Simionescu, Olga; Popescu, Bogdan Ovidiu; Costache, Mariana; Manole, Emilia; Spulber, Stefan; Gherghiceanu, Mihaela; Blum, Andreas

    2012-06-01

    The aetiology of seborrheic keratoses (SK), the most common benign epithelial tumours, and any relationship with malignancy are not yet known. As a protective anti-cancer mechanism, apoptosis reflects cellular loss as a reaction to proliferative activity. The objective of this study was to quantify apoptosis in different SK types (acanthotic, hyperkeratotic, reticulated, irritated and clonal) and correlate the dermoscopic picture with apoptosis rate. After a qualitative and quantitative analysis of dermoscopic images, we defined a new quantitative dermoscopic score (C3V2F, crypts, millia cysts, colours, hairpin vessels, irregular vessels, fissures) from 0 to 22, which enabled us to establish cut-offs correlating with apoptosis rates. All five SK forms were associated with lower apoptosis rates compared with normal skin. A C3V2F score >10 and greater number of crypts and colours reflected a higher apoptosis rate, which implies a benign character of evolution. In contrast, the presence of irregular vessels on more than 50% of the lesion surface implied a lower rate of apoptosis and probably associated with a risk of malignant transformation. On the basis of dermoscopic information, we used multiple regression to establish a model for estimating the rate of apoptosis with a 0.7 prediction interval (approximately 1S.D.). The new C3V2F score could be valuable for the clinical evaluation of possible SK prognosis and decisions regarding excision. PMID:22404841

  10. Intranuclear Actin Regulates Osteogenesis

    PubMed Central

    Sen, Buer; Xie, Zhihui; Uzer, Gunes; Thompson, William R.; Styner, Maya; Wu, Xin; Rubin, Janet

    2016-01-01

    Depolymerization of the actin cytoskeleton induces nuclear trafficking of regulatory proteins and global effects on gene transcription. We here show that in mesenchymal stem cells (MSCs), cytochalasin D treatment causes rapid cofilin-/importin-9-dependent transfer of G-actin into the nucleus. The continued presence of intranuclear actin, which forms rod-like structures that stain with phalloidin, is associated with induction of robust expression of the osteogenic genes osterix and osteocalcin in a Runx2-dependent manner, and leads to acquisition of osteogenic phenotype. Adipogenic differentiation also occurs, but to a lesser degree. Intranuclear actin leads to nuclear export of Yes-associated protein (YAP); maintenance of nuclear YAP inhibits Runx2 initiation of osteogenesis. Injection of cytochalasin into the tibial marrow space of live mice results in abundant bone formation within the space of 1 week. In sum, increased intranuclear actin forces MSC into osteogenic lineage through controlling Runx2 activity; this process may be useful for clinical objectives of forming bone. PMID:26140478

  11. Amplification of actin polymerization forces

    PubMed Central

    Dmitrieff, Serge; Nédélec, François

    2016-01-01

    The actin cytoskeleton drives many essential processes in vivo, using molecular motors and actin assembly as force generators. We discuss here the propagation of forces caused by actin polymerization, highlighting simple configurations where the force developed by the network can exceed the sum of the polymerization forces from all filaments. PMID:27002174

  12. In vivo reflectance confocal microscopy characterization of field-directed 5-fluorouracil 0.5%/salicylic acid 10% in actinic keratosis.

    PubMed

    Ulrich, Martina; Alarcon, Ivette; Malvehy, Josep; Puig, Susana

    2015-01-01

    Actinic keratosis (AK), a frequently diagnosed cutaneous neoplasm in individuals with chronic sun exposure or fair skin, is a risk factor for squamous cell carcinoma. AK presents as clinically visible lesions and/or as subclinical lesions where an entire field of area (field cancerization) contains lesions of various grades. The diagnosis and surveillance of subclinical AK is challenging. We report a new AK management approach, including subclinical AK, with noninvasive in vivo reflectance confocal microscopy (RCM) monitoring of field-directed topical 5-fluorouracil 0.5%/salicylic acid 10.0% (5-FU/SA; currently approved for single lesions). In this case series, eight patients with primarily recurrent, multiple AKs received ≤ 6 weeks of field-directed 5-FU/SA; complete clearance of clinical/subclinical AKs on various body areas was shown in most patients using RCM. RCM facilitated the detection/characterization of subclinical AKs in the setting of field cancerization. Topical field-directed 5-FU/SA monitored with RCM is a promising management approach for subclinical AKs.

  13. Control of actin-based motility through localized actin binding.

    PubMed

    Banigan, Edward J; Lee, Kun-Chun; Liu, Andrea J

    2013-12-01

    A wide variety of cell biological and biomimetic systems use actin polymerization to drive motility. It has been suggested that an object such as a bacterium can propel itself by self-assembling a high concentration of actin behind it, if it is repelled by actin. However, it is also known that it is essential for the moving object to bind actin. Therefore, a key question is how the actin tail can propel an object when it both binds and repels the object. We present a physically consistent Brownian dynamics model for actin-based motility that includes the minimal components of the dendritic nucleation model and allows for both attractive and repulsive interactions between actin and a moveable disc. We find that the concentration gradient of filamentous actin generated by polymerization is sufficient to propel the object, even with moderately strong binding interactions. Additionally, actin binding can act as a biophysical cap, and may directly control motility through modulation of network growth. Overall, this mechanism is robust in that it can drive motility against a load up to a stall pressure that depends on the Young's modulus of the actin network and can explain several aspects of actin-based motility.

  14. Guardians of the actin monomer.

    PubMed

    Xue, Bo; Robinson, Robert C

    2013-01-01

    Actin is a universal force provider in eukaryotic cells. Biological processes harness the pressure generated from actin polymerization through dictating the time, place and direction of filament growth. As such, polymerization is initiated and maintained via tightly controlled filament nucleation and elongation machineries. Biological systems integrate force into their activities through recruiting and activating these machineries. In order that actin function as a common force generating polymerization motor, cells must maintain a pool of active, polymerization-ready monomeric actin, and minimize extemporaneous polymerization. Maintenance of the active monomeric actin pool requires the recycling of actin filaments, through depolymerization, nucleotide exchange and reloading of the polymerization machineries, while the levels of monomers are constantly monitored and supplemented, when needed, via the access of a reserve pool of monomers and through gene expression. Throughout its monomeric life, actin needs to be protected against gratuitous nucleation events. Here, we review the proteins that act as custodians of monomeric actin. We estimate their levels on a tissue scale, and calculate the implied concentrations of each actin complex based on reported binding affinities. These estimations predict that monomeric actin is rarely, if ever, alone. Thus, the guardians keep the volatility of actin in check, so that its explosive power is only released in the controlled environments of the nucleation and polymerization machineries. PMID:24268205

  15. Autophosphorylation at Thr279 of Entamoeba histolytica atypical kinase EhAK1 is required for activity and regulation of erythrophagocytosis

    PubMed Central

    Mansuri, M Shahid; Babuta, Mrigya; Ali, Mohammad Sabir; Bharadwaj, Ravi; jhingan, Gagan Deep; Gourinath, Samudrala; Bhattacharya, Sudha; Bhattacharya, Alok

    2016-01-01

    Phagocytosis plays a key role in survival and pathogenicity of Entamoeba histolytica. We have recently demonstrated that an atypical kinase EhAK1 is involved in phagocytosis in this parasite. It is recruited to the phagocytic cups through interaction with EhCaBP1. EhAK1 manipulates actin dynamics by multiple mechanisms including phosphorylation of G-actin. Biochemical analysis showed that EhAK1 is a serine/threonine kinase with broad ion specificity and undergoes multiple trans-autophosphorylation. Three autophosphorylation sites were identified by mass spectrometry. Out of these Thr279 appears to be involved in both autophosphorylation as well as substrate phosphorylation. Over expression of the mutant Thr279A inhibited erythrophagocytosis showing dominant negative phenotype. Multiple alignments of different kinases including alpha kinases displayed conserved binding sites that are thought to be important for function of the protein. Mutation studies demonstrated the importance of some of these binding sites in kinase activity. Binding studies with fluorescent-ATP analogs supported our prediction regarding ATP binding site based on sequence alignment. In conclusion, EhAK1 has multiple regulatory features and enrichment of EhAK1 at the site of phagocytosis stimulates trans-autophosphorylation reaction that increases kinase activity resulting in enhanced actin dynamics and phagocytosis. Some of the properties of EhAK1 are similar to that seen in alpha kinases. PMID:26739245

  16. Actin stress in cell reprogramming

    PubMed Central

    Guo, Jun; Wang, Yuexiu; Sachs, Frederick; Meng, Fanjie

    2014-01-01

    Cell mechanics plays a role in stem cell reprogramming and differentiation. To understand this process better, we created a genetically encoded optical probe, named actin–cpstFRET–actin (AcpA), to report forces in actin in living cells in real time. We showed that stemness was associated with increased force in actin. We reprogrammed HEK-293 cells into stem-like cells using no transcription factors but simply by softening the substrate. However, Madin-Darby canine kidney (MDCK) cell reprogramming required, in addition to a soft substrate, Harvey rat sarcoma viral oncogene homolog expression. Replating the stem-like cells on glass led to redifferentiation and reduced force in actin. The actin force probe was a FRET sensor, called cpstFRET (circularly permuted stretch sensitive FRET), flanked by g-actin subunits. The labeled actin expressed efficiently in HEK, MDCK, 3T3, and bovine aortic endothelial cells and in multiple stable cell lines created from those cells. The viability of the cell lines demonstrated that labeled actin did not significantly affect cell physiology. The labeled actin distribution was similar to that observed with GFP-tagged actin. We also examined the stress in the actin cross-linker actinin. Actinin force was not always correlated with actin force, emphasizing the need for addressing protein specificity when discussing forces. Because actin is a primary structural protein in animal cells, understanding its force distribution is central to understanding animal cell physiology and the many linked reactions such as stress-induced gene expression. This new probe permits measuring actin forces in a wide range of experiments on preparations ranging from isolated proteins to transgenic animals. PMID:25422450

  17. Actin dynamics in living mammalian cells.

    PubMed

    Ballestrem, C; Wehrle-Haller, B; Imhof, B A

    1998-06-01

    The actin cytoskeleton maintains the cellular architecture and mediates cell movements. To explore actin cytoskeletal dynamics, the enhanced green fluorescent protein (EGFP) was fused to human &bgr ;-actin. The fusion protein was incorporated into actin fibers which became depolymerized upon cytochalasin B treatment. This functional EGFP-actin construct enabled observation of the actin cytoskeleton in living cells by time lapse fluorescence microscopy. Stable expression of the construct was obtained in mammalian cell lines of different tissue origins. In stationary cells, actin rich, ring-like structured 'actin clouds' were observed in addition to stress fibers. These ruffle-like structures were found to be involved in the reorganization of the actin cytoskeleton. In migratory cells, EGFP-actin was found in the advancing lamellipodium. Immobile actin spots developed in the lamellipodium and thin actin fibers formed parallel to the leading edge. Thus EGFP-actin expressed in living cells unveiled structures involved in the dynamics of the actin cytoskeleton.

  18. What is the role of field-directed therapy in the treatment of actinic keratosis? Part 2: Commonly used field-directed and lesion-directed therapies.

    PubMed

    Berman, Brian; Cohen, David E; Amini, Sadegh

    2012-06-01

    Actinic keratosis (AK) constitutes the initial epidermal lesion in a disease continuum that may potentially progress to invasive squamous cell carcinoma (SCC). A number of treatment options are available to clear lesions and thus reduce the risk for progression to SCC. Field-directed therapies are primarily used to clear multiple AKs and subclinical lesions. Part 1 of this review explaining the role of field-directed therapy for the treatment of AK discussed the unmet needs with current therapies and the investigational agents that are being developed to fill treatment gaps. Part 2 will mainly focus on field-directed therapies that currently are available for AK, such as resurfacing procedures, patient-administered topical therapy, and photodynamic therapy (PDT), as well as lesion-directed therapy, which is used to clear discrete lesions in relatively small numbers. PMID:22838095

  19. Formin' actin in the nucleus.

    PubMed

    Baarlink, Christian; Grosse, Robert

    2014-01-01

    Many if not most proteins can, under certain conditions, change cellular compartments, such as, for example, shuttling from the cytoplasm to the nucleus. Thus, many proteins may exert functions in various and very different subcellular locations, depending on the signaling context. A large amount of actin regulatory proteins has been detected in the mammalian cell nucleus, although their potential roles are much debated and are just beginning to emerge. Recently, members of the formin family of actin nucleators were also reported to dynamically localize to the nuclear environment. Here we discuss our findings that specific diaphanous-related formins can promote nuclear actin assembly in a signal-dependent manner.

  20. MAL Daylight Photodynamic Therapy for Actinic Keratosis: Clinical and Imaging Evaluation by 3D Camera.

    PubMed

    Cantisani, Carmen; Paolino, Giovanni; Pellacani, Giovanni; Didona, Dario; Scarno, Marco; Faina, Valentina; Gobello, Tommaso; Calvieri, Stefano

    2016-07-11

    Non-melanoma skin cancer is the most common skin cancer with an incidence that varies widely worldwide. Among them, actinic keratosis (AK), considered by some authors as in situ squamous cell carcinoma (SCC), are the most common and reflect an abnormal multistep skin cell development due to the chronic ultraviolet (UV) light exposure. No ideal treatment exists, but the potential risk of their development in a more invasive form requires prompt treatment. As patients usually present with multiple AK on fields of actinic damage, there is a need for effective, safe, simple and short treatments which allow the treatment of large areas. To achieve this, daylight photodynamic therapy (DL-PDT) is an innovative treatment for multiple mild actinic keratosis, well tolerated by patients. Patients allocated to the PDT unit, affected by multiple mild-moderate and severe actinic keratosis on sun-exposed areas treated with DL-PDT, were clinically evaluated at baseline and every three months with an Antera 3D, Miravex(©) camera. Clinical and 3D images were performed at each clinical check almost every three months. In this retrospective study, 331 patients (56.7% male, 43.3% female) were treated with DL-PDT. We observed a full clearance in more than two-thirds of patients with one or two treatments. Different responses depend on the number of lesions and on their severity; for patients with 1-3 lesions and with grade I or II AK, a full clearance was reached in 85% of cases with a maximum of two treatments. DL-PDT in general improved skin tone and erased sun damage. Evaluating each Antera 3D images, hemoglobin concentration and pigmentation, a skin color and tone improvement in 310 patients was observed. DL-PDT appears as a promising, effective, simple, tolerable and practical treatment for actinic damage associated with AK, and even treatment of large areas can be with little or no pain. The 3D imaging allowed for quantifying in real time the aesthetic benefits of DL

  1. MAL Daylight Photodynamic Therapy for Actinic Keratosis: Clinical and Imaging Evaluation by 3D Camera

    PubMed Central

    Cantisani, Carmen; Paolino, Giovanni; Pellacani, Giovanni; Didona, Dario; Scarno, Marco; Faina, Valentina; Gobello, Tommaso; Calvieri, Stefano

    2016-01-01

    Non-melanoma skin cancer is the most common skin cancer with an incidence that varies widely worldwide. Among them, actinic keratosis (AK), considered by some authors as in situ squamous cell carcinoma (SCC), are the most common and reflect an abnormal multistep skin cell development due to the chronic ultraviolet (UV) light exposure. No ideal treatment exists, but the potential risk of their development in a more invasive form requires prompt treatment. As patients usually present with multiple AK on fields of actinic damage, there is a need for effective, safe, simple and short treatments which allow the treatment of large areas. To achieve this, daylight photodynamic therapy (DL-PDT) is an innovative treatment for multiple mild actinic keratosis, well tolerated by patients. Patients allocated to the PDT unit, affected by multiple mild−moderate and severe actinic keratosis on sun-exposed areas treated with DL-PDT, were clinically evaluated at baseline and every three months with an Antera 3D, Miravex© camera. Clinical and 3D images were performed at each clinical check almost every three months. In this retrospective study, 331 patients (56.7% male, 43.3% female) were treated with DL-PDT. We observed a full clearance in more than two-thirds of patients with one or two treatments. Different responses depend on the number of lesions and on their severity; for patients with 1–3 lesions and with grade I or II AK, a full clearance was reached in 85% of cases with a maximum of two treatments. DL-PDT in general improved skin tone and erased sun damage. Evaluating each Antera 3D images, hemoglobin concentration and pigmentation, a skin color and tone improvement in 310 patients was observed. DL-PDT appears as a promising, effective, simple, tolerable and practical treatment for actinic damage associated with AK, and even treatment of large areas can be with little or no pain. The 3D imaging allowed for quantifying in real time the aesthetic benefits of DL

  2. 46 CFR 7.170 - Alaska Peninsula, AK to Aleutian Islands, AK.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 1 2011-10-01 2011-10-01 false Alaska Peninsula, AK to Aleutian Islands, AK. 7.170... BOUNDARY LINES Alaska § 7.170 Alaska Peninsula, AK to Aleutian Islands, AK. (a) A line drawn from the southernmost extremity of Cape Kumlium to the westernmost extremity of Nakchamik Island; thence to...

  3. 46 CFR 7.165 - Kenai Peninsula, AK to Kodiak Island, AK.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 1 2010-10-01 2010-10-01 false Kenai Peninsula, AK to Kodiak Island, AK. 7.165 Section... BOUNDARY LINES Alaska § 7.165 Kenai Peninsula, AK to Kodiak Island, AK. (a) A line drawn from the southernmost extremity of Kenai Peninsula at longitude 151°44.0′ W. to East Amatuli Island Light; thence to...

  4. 46 CFR 7.170 - Alaska Peninsula, AK to Aleutian Islands, AK.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 1 2012-10-01 2012-10-01 false Alaska Peninsula, AK to Aleutian Islands, AK. 7.170... BOUNDARY LINES Alaska § 7.170 Alaska Peninsula, AK to Aleutian Islands, AK. (a) A line drawn from the southernmost extremity of Cape Kumlium to the westernmost extremity of Nakchamik Island; thence to...

  5. 46 CFR 7.165 - Kenai Peninsula, AK to Kodiak Island, AK.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 1 2014-10-01 2014-10-01 false Kenai Peninsula, AK to Kodiak Island, AK. 7.165 Section... BOUNDARY LINES Alaska § 7.165 Kenai Peninsula, AK to Kodiak Island, AK. (a) A line drawn from the southernmost extremity of Kenai Peninsula at longitude 151°44.0′ W. to East Amatuli Island Light; thence to...

  6. 46 CFR 7.170 - Alaska Peninsula, AK to Aleutian Islands, AK.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 1 2014-10-01 2014-10-01 false Alaska Peninsula, AK to Aleutian Islands, AK. 7.170... BOUNDARY LINES Alaska § 7.170 Alaska Peninsula, AK to Aleutian Islands, AK. (a) A line drawn from the southernmost extremity of Cape Kumlium to the westernmost extremity of Nakchamik Island; thence to...

  7. 46 CFR 7.170 - Alaska Peninsula, AK to Aleutian Islands, AK.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 1 2010-10-01 2010-10-01 false Alaska Peninsula, AK to Aleutian Islands, AK. 7.170... BOUNDARY LINES Alaska § 7.170 Alaska Peninsula, AK to Aleutian Islands, AK. (a) A line drawn from the southernmost extremity of Cape Kumlium to the westernmost extremity of Nakchamik Island; thence to...

  8. 46 CFR 7.165 - Kenai Peninsula, AK to Kodiak Island, AK.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 1 2012-10-01 2012-10-01 false Kenai Peninsula, AK to Kodiak Island, AK. 7.165 Section... BOUNDARY LINES Alaska § 7.165 Kenai Peninsula, AK to Kodiak Island, AK. (a) A line drawn from the southernmost extremity of Kenai Peninsula at longitude 151°44.0′ W. to East Amatuli Island Light; thence to...

  9. 46 CFR 7.165 - Kenai Peninsula, AK to Kodiak Island, AK.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 1 2011-10-01 2011-10-01 false Kenai Peninsula, AK to Kodiak Island, AK. 7.165 Section... BOUNDARY LINES Alaska § 7.165 Kenai Peninsula, AK to Kodiak Island, AK. (a) A line drawn from the southernmost extremity of Kenai Peninsula at longitude 151°44.0′ W. to East Amatuli Island Light; thence to...

  10. 46 CFR 7.170 - Alaska Peninsula, AK to Aleutian Islands, AK.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 1 2013-10-01 2013-10-01 false Alaska Peninsula, AK to Aleutian Islands, AK. 7.170... BOUNDARY LINES Alaska § 7.170 Alaska Peninsula, AK to Aleutian Islands, AK. (a) A line drawn from the southernmost extremity of Cape Kumlium to the westernmost extremity of Nakchamik Island; thence to...

  11. 46 CFR 7.165 - Kenai Peninsula, AK to Kodiak Island, AK.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 1 2013-10-01 2013-10-01 false Kenai Peninsula, AK to Kodiak Island, AK. 7.165 Section... BOUNDARY LINES Alaska § 7.165 Kenai Peninsula, AK to Kodiak Island, AK. (a) A line drawn from the southernmost extremity of Kenai Peninsula at longitude 151°44.0′ W. to East Amatuli Island Light; thence to...

  12. Regulation of an Actin Spring

    NASA Astrophysics Data System (ADS)

    Tam, Barney; Shin, Jennifer; Brau, Ricardo; Lang, Matthew; Mahadevan, L.; Matsudaira, Paul

    2006-03-01

    To produce motion, cells rely on the conversion of potential energy into mechanical work. One such example is the dramatic process involving the acrosome reaction of Limulus sperm, whereby a 60 μm-long bundle of actin filaments straightens from a coiled conformation to extend out of the cell in five seconds. This cellular engine and the motion it produces represent a third type of actin-based motility fundamentally different from polymerization or myosin-driven processes. The motive force for this extension originates from stored elastic energy in the overtwisted, pre-formed coil---much like a compressed mechanical spring. When the actin bundle untwists, this energy is converted to mechanical work powering the extension. We report on experiments probing the regulation of this actin spring by extracellular calcium. We find that extracellular calcium needs to be present for the spring to activate, and that calcium regulates the velocity of the extension.

  13. Chemotaxis and Actin Oscillations

    NASA Astrophysics Data System (ADS)

    Bodenschatz, Eberhard; Hsu, Hsin-Fang; Negrete, Jose; Beta, Carsten; Pumir, Alain; Gholami, Azam; Tarantola, Marco; Westendorf, Christian; Zykov, Vladimir

    Recently, self-oscillations of the cytoskeletal actin have been observed in Dictyostelium, a model system for studying chemotaxis. Here we report experimental results on the self-oscillation mechanism and the role of regulatory proteins and myosin II. We stimulate cells rapidly and periodically by using photo un-caging of the chemoattractant in a micro-fluidic device and measured the cellular responses. We found that the response amplitude grows with stimulation strength only in a very narrow region of stimulation, after which the response amplitude reaches a plateau. Moreover, the frequency-response is not constant but rather varies with the strength of external stimuli. To understand the underlying mechanism, we analyzed the polymerization and de-polymerization time in the single cell level. Despite of the large cell-to-cell variability, we found that the polymerization time is independent of external stimuli and the de-polymerization time is prolonged as the stimulation strength increases. Our conclusions will be summarized and the role of noise in the signaling network will be discussed. German Science Foundation CRC 937.

  14. Three's company: the fission yeast actin cytoskeleton.

    PubMed

    Kovar, David R; Sirotkin, Vladimir; Lord, Matthew

    2011-03-01

    How the actin cytoskeleton assembles into different structures to drive diverse cellular processes is a fundamental cell biological question. In addition to orchestrating the appropriate combination of regulators and actin-binding proteins, different actin-based structures must insulate themselves from one another to maintain specificity within a crowded cytoplasm. Actin specification is particularly challenging in complex eukaryotes where a multitude of protein isoforms and actin structures operate within the same cell. Fission yeast Schizosaccharomyces pombe possesses a single actin isoform that functions in three distinct structures throughout the cell cycle. In this review we explore recent studies in fission yeast that help unravel how different actin structures operate in cells.

  15. Structural insights into de novo actin polymerization

    PubMed Central

    Dominguez, Roberto

    2010-01-01

    Summary Many cellular functions depend on rapid and localized actin polymerization/depolymerization. Yet, the de novo polymerization of actin in cells is kinetically unfavorable because of the instability of polymerization intermediates (small actin oligomers) and the actions of actin monomer binding proteins. Cells use filament nucleation and elongation factors to initiate and sustain polymerization. Structural biology is beginning to shed light on the diverse mechanisms by which these unrelated proteins initiate polymerization, undergo regulation, and mediate the transition of monomeric actin onto actin filaments. A prominent role is played by the W domain, which in some of these proteins occurs in tandem repeats that recruit multiple actin subunits. Pro-rich regions are also abundant and mediate the binding of profilin-actin complexes, which are the main source of polymerization competent actin in cells. Filament nucleation and elongation factors frequently interact with Rho family GTPases, which relay signals from membrane receptors to regulate actin cytoskeleton remodeling. PMID:20096561

  16. Polymerization of Actin from Maize Pollen.

    PubMed Central

    Yen, L. F.; Liu, X.; Cai, S.

    1995-01-01

    Here we describe the in vitro polymerization of actin from maize (Zea mays) pollen. The purified actin from maize pollen reported in our previous paper (X. Liu, L.F. Yen [1992] Plant Physiol 99: 1151-1155) is biologically active. In the presence of ATP, KCl, and MgCl2 the purified pollen actin polymerized into filaments. During polymerization the spectra of absorbance at 232 nm increased gradually. Polymerization of pollen actin was evidently accompanied by an increase in viscosity of the pollen actin solution. Also, the specific viscosity of pollen F-actin increased in a concentration-dependent manner. The ultraviolet difference spectrum of pollen actin is very similar to that of rabbit muscle actin. The activity of myosin ATPase from rabbit muscle was activated 7-fold by the polymerized pollen actin (F-actin). The actin filaments were visualized under the electron microscope as doubly wound strands of 7 nm diameter. If cytochalasin B was added before staining, no actin filaments were observed. When actin filaments were treated with rabbit heavy meromyosin, the actin filaments were decorated with an arrowhead structure. These results imply that there is much similarity between pollen and muscle actin. PMID:12228343

  17. Cofilin-mediated actin dynamics promotes actin bundle formation during Drosophila bristle development

    PubMed Central

    Wu, Jing; Wang, Heng; Guo, Xuan; Chen, Jiong

    2016-01-01

    The actin bundle is an array of linear actin filaments cross-linked by actin-bundling proteins, but its assembly and dynamics are not as well understood as those of the branched actin network. Here we used the Drosophila bristle as a model system to study actin bundle formation. We found that cofilin, a major actin disassembly factor of the branched actin network, promotes the formation and positioning of actin bundles in the developing bristles. Loss of function of cofilin or AIP1, a cofactor of cofilin, each resulted in increased F-actin levels and severe defects in actin bundle organization, with the defects from cofilin deficiency being more severe. Further analyses revealed that cofilin likely regulates actin bundle formation and positioning by the following means. First, cofilin promotes a large G-actin pool both locally and globally, likely ensuring rapid actin polymerization for bundle initiation and growth. Second, cofilin limits the size of a nonbundled actin-myosin network to regulate the positioning of actin bundles. Third, cofilin prevents incorrect assembly of branched and myosin-associated actin filament into bundles. Together these results demonstrate that the interaction between the dynamic dendritic actin network and the assembling actin bundles is critical for actin bundle formation and needs to be closely regulated. PMID:27385345

  18. Cofilin-mediated actin dynamics promotes actin bundle formation during Drosophila bristle development.

    PubMed

    Wu, Jing; Wang, Heng; Guo, Xuan; Chen, Jiong

    2016-08-15

    The actin bundle is an array of linear actin filaments cross-linked by actin-bundling proteins, but its assembly and dynamics are not as well understood as those of the branched actin network. Here we used the Drosophila bristle as a model system to study actin bundle formation. We found that cofilin, a major actin disassembly factor of the branched actin network, promotes the formation and positioning of actin bundles in the developing bristles. Loss of function of cofilin or AIP1, a cofactor of cofilin, each resulted in increased F-actin levels and severe defects in actin bundle organization, with the defects from cofilin deficiency being more severe. Further analyses revealed that cofilin likely regulates actin bundle formation and positioning by the following means. First, cofilin promotes a large G-actin pool both locally and globally, likely ensuring rapid actin polymerization for bundle initiation and growth. Second, cofilin limits the size of a nonbundled actin-myosin network to regulate the positioning of actin bundles. Third, cofilin prevents incorrect assembly of branched and myosin-associated actin filament into bundles. Together these results demonstrate that the interaction between the dynamic dendritic actin network and the assembling actin bundles is critical for actin bundle formation and needs to be closely regulated.

  19. Boolean gates on actin filaments

    NASA Astrophysics Data System (ADS)

    Siccardi, Stefano; Tuszynski, Jack A.; Adamatzky, Andrew

    2016-01-01

    Actin is a globular protein which forms long polar filaments in the eukaryotic cytoskeleton. Actin networks play a key role in cell mechanics and cell motility. They have also been implicated in information transmission and processing, memory and learning in neuronal cells. The actin filaments have been shown to support propagation of voltage pulses. Here we apply a coupled nonlinear transmission line model of actin filaments to study interactions between voltage pulses. To represent digital information we assign a logical TRUTH value to the presence of a voltage pulse in a given location of the actin filament, and FALSE to the pulse's absence, so that information flows along the filament with pulse transmission. When two pulses, representing Boolean values of input variables, interact, then they can facilitate or inhibit further propagation of each other. We explore this phenomenon to construct Boolean logical gates and a one-bit half-adder with interacting voltage pulses. We discuss implications of these findings on cellular process and technological applications.

  20. Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography

    NASA Technical Reports Server (NTRS)

    Hu, S.; Brady, S. R.; Kovar, D. R.; Staiger, C. J.; Clark, G. B.; Roux, S. J.; Muday, G. K.

    2000-01-01

    Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.

  1. In β-actin knockouts, epigenetic reprogramming and rDNA transcription inactivation lead to growth and proliferation defects.

    PubMed

    Almuzzaini, Bader; Sarshad, Aishe A; Rahmanto, Aldwin S; Hansson, Magnus L; Von Euler, Anne; Sangfelt, Olle; Visa, Neus; Farrants, Ann-Kristin Östlund; Percipalle, Piergiorgio

    2016-08-01

    Actin and nuclear myosin 1 (NM1) are regulators of transcription and chromatin organization. Using a genome-wide approach, we report here that β-actin binds intergenic and genic regions across the mammalian genome, associated with both protein-coding and rRNA genes. Within the rDNA, the distribution of β-actin correlated with NM1 and the other subunits of the B-WICH complex, WSTF and SNF2h. In β-actin(-/-) mouse embryonic fibroblasts (MEFs), we found that rRNA synthesis levels decreased concomitantly with drops in RNA polymerase I (Pol I) and NM1 occupancies across the rRNA gene. Reintroduction of wild-type β-actin, in contrast to mutated forms with polymerization defects, efficiently rescued rRNA synthesis underscoring the direct role for a polymerization-competent form of β-actin in Pol I transcription. The rRNA synthesis defects in the β-actin(-/-) MEFs are a consequence of epigenetic reprogramming with up-regulation of the repressive mark H3K4me1 (monomethylation of lys4 on histone H3) and enhanced chromatin compaction at promoter-proximal enhancer (T0 sequence), which disturb binding of the transcription factor TTF1. We propose a novel genome-wide mechanism where the polymerase-associated β-actin synergizes with NM1 to coordinate permissive chromatin with Pol I transcription, cell growth, and proliferation.-Almuzzaini, B., Sarshad, A. A. , Rahmanto, A. S., Hansson, M. L., Von Euler, A., Sangfelt, O., Visa, N., Farrants, A.-K. Ö., Percipalle, P. In β-actin knockouts, epigenetic reprogramming and rDNA transcription inactivation lead to growth and proliferation defects.

  2. An Investigator-initiated Study to Assess the Safety and Efficacy of Ingenol Mebutate 0.05% Gel When Used After Cryosurgery in the Treatment of Hypertrophic Actinic Keratosis on Dorsal Hands

    PubMed Central

    Hashim, Peter W.; Nia, John K.; Singer, Skylar

    2016-01-01

    Objectives: To evaluate the safety and efficacy of ingenol mebutate 0.05% gel after cryosurgery versus cryosurgery alone for the treatment of hypertrophic and nonhypertrophic actinic keratosis on the dorsal hands. Design: Investigator-blinded split arm study. Setting: Academic institution. Participants: Sixteen subjects with actinic keratoses on dorsal hands. Results: There was a mean reduction in the number of hypertrophic actinic keratosis lesions adjusted for baseline in ingenol mebutate-treated versus control group of -4.3 versus -2.8, respectively. There was a mean reduction in the number of non-hypertrophic actinic keratosis lesions in the ingenol mebutate-treated versus control group of -3.8 versus -0.3. Conclusion: A statistically significant and clinically meaningful difference in response was demonstrated in favor of ingenol mebutate-treated hands versus controls. No significant increase in local skin responses was noted when applying ingenol mebutate 0.05% gel on the same day as cryosurgery. Trial registry: ClinicalTrials.gov, NCT02251652. PMID:27672408

  3. An Investigator-initiated Study to Assess the Safety and Efficacy of Ingenol Mebutate 0.05% Gel When Used After Cryosurgery in the Treatment of Hypertrophic Actinic Keratosis on Dorsal Hands

    PubMed Central

    Hashim, Peter W.; Nia, John K.; Singer, Skylar

    2016-01-01

    Objectives: To evaluate the safety and efficacy of ingenol mebutate 0.05% gel after cryosurgery versus cryosurgery alone for the treatment of hypertrophic and nonhypertrophic actinic keratosis on the dorsal hands. Design: Investigator-blinded split arm study. Setting: Academic institution. Participants: Sixteen subjects with actinic keratoses on dorsal hands. Results: There was a mean reduction in the number of hypertrophic actinic keratosis lesions adjusted for baseline in ingenol mebutate-treated versus control group of -4.3 versus -2.8, respectively. There was a mean reduction in the number of non-hypertrophic actinic keratosis lesions in the ingenol mebutate-treated versus control group of -3.8 versus -0.3. Conclusion: A statistically significant and clinically meaningful difference in response was demonstrated in favor of ingenol mebutate-treated hands versus controls. No significant increase in local skin responses was noted when applying ingenol mebutate 0.05% gel on the same day as cryosurgery. Trial registry: ClinicalTrials.gov, NCT02251652.

  4. Bacterial Actins? An Evolutionary Perspective

    NASA Technical Reports Server (NTRS)

    Doolittle, Russell F.; York, Amanda L.

    2003-01-01

    According to the conventional wisdom, the existence of a cytoskeleton in eukaryotes and its absence in prokaryotes constitute a fundamental divide between the two domains of life. An integral part of the dogma is that a cytoskeleton enabled an early eukaryote to feed upon prokaryotes, a consequence of which was the occasional endosymbiosis and the eventual evolution of organelles. Two recent papers present compelling evidence that actin, one of the principal components of a cytoskeleton, has a homolog in Bacteria that behaves in many ways like eukaryotic actin. Sequence comparisons reveml that eukaryotic actin and the bacterial homolog (mreB protein), unlike many other proteins common to eukaryotes and Bacteria, have very different and more highly extended evolutionary histories.

  5. Fascin regulates nuclear actin during Drosophila oogenesis.

    PubMed

    Kelpsch, Daniel J; Groen, Christopher M; Fagan, Tiffany N; Sudhir, Sweta; Tootle, Tina L

    2016-10-01

    Drosophila oogenesis provides a developmental system with which to study nuclear actin. During Stages 5-9, nuclear actin levels are high in the oocyte and exhibit variation within the nurse cells. Cofilin and Profilin, which regulate the nuclear import and export of actin, also localize to the nuclei. Expression of GFP-tagged Actin results in nuclear actin rod formation. These findings indicate that nuclear actin must be tightly regulated during oogenesis. One factor mediating this regulation is Fascin. Overexpression of Fascin enhances nuclear GFP-Actin rod formation, and Fascin colocalizes with the rods. Loss of Fascin reduces, whereas overexpression of Fascin increases, the frequency of nurse cells with high levels of nuclear actin, but neither alters the overall nuclear level of actin within the ovary. These data suggest that Fascin regulates the ability of specific cells to accumulate nuclear actin. Evidence indicates that Fascin positively regulates nuclear actin through Cofilin. Loss of Fascin results in decreased nuclear Cofilin. In addition, Fascin and Cofilin genetically interact, as double heterozygotes exhibit a reduction in the number of nurse cells with high nuclear actin levels. These findings are likely applicable beyond Drosophila follicle development, as the localization and functions of Fascin and the mechanisms regulating nuclear actin are widely conserved.

  6. Fascin regulates nuclear actin during Drosophila oogenesis.

    PubMed

    Kelpsch, Daniel J; Groen, Christopher M; Fagan, Tiffany N; Sudhir, Sweta; Tootle, Tina L

    2016-10-01

    Drosophila oogenesis provides a developmental system with which to study nuclear actin. During Stages 5-9, nuclear actin levels are high in the oocyte and exhibit variation within the nurse cells. Cofilin and Profilin, which regulate the nuclear import and export of actin, also localize to the nuclei. Expression of GFP-tagged Actin results in nuclear actin rod formation. These findings indicate that nuclear actin must be tightly regulated during oogenesis. One factor mediating this regulation is Fascin. Overexpression of Fascin enhances nuclear GFP-Actin rod formation, and Fascin colocalizes with the rods. Loss of Fascin reduces, whereas overexpression of Fascin increases, the frequency of nurse cells with high levels of nuclear actin, but neither alters the overall nuclear level of actin within the ovary. These data suggest that Fascin regulates the ability of specific cells to accumulate nuclear actin. Evidence indicates that Fascin positively regulates nuclear actin through Cofilin. Loss of Fascin results in decreased nuclear Cofilin. In addition, Fascin and Cofilin genetically interact, as double heterozygotes exhibit a reduction in the number of nurse cells with high nuclear actin levels. These findings are likely applicable beyond Drosophila follicle development, as the localization and functions of Fascin and the mechanisms regulating nuclear actin are widely conserved. PMID:27535426

  7. Assessment of local skin reactions with a sequential regimen of cryosurgery followed by ingenol mebutate gel, 0.015%, in patients with actinic keratosis.

    PubMed

    Goldenberg, Gary; Berman, Brian

    2015-01-01

    Lesion-directed and field-directed therapies are used to treat actinic keratosis (AK). Therapeutic approaches that combine both types of therapies may improve the successful elimination of AKs. A randomized, double-blind, vehicle-controlled study evaluated the safety, tolerability, and efficacy of topical field treatment with ingenol mebutate gel, 0.015%, after cryosurgery to AKs on the face and scalp. Patients with 4-8 visible discrete AKs in a 25-cm(2) contiguous area received cryosurgery of all AKs at baseline. After a 3-week healing period, patients applied ingenol mebutate gel, 0.015%, or vehicle gel once daily for 3 consecutive days to the treatment area. The incidence, severity, and time course of the development and resolution of local skin reactions were measured from baseline to week 11. Local skin reactions peaked shortly after completion of ingenol mebutate treatment and generally resolved within 2 weeks. The mean (95% confidence interval) composite score (maximum range, 0-24) for these reactions was higher in patients with treatment of AKs on the face, 9.3 (8.5-10.1), as compared with the scalp, 5.8 (4.3-7.4). Erythema and flaking/scaling were the major contributors to the composite local skin reaction score. These results show that local skin reactions associated with ingenol mebutate treatment of the face or scalp are well tolerated after recent cryosurgery. PMID:25565875

  8. Assessment of local skin reactions with a sequential regimen of cryosurgery followed by ingenol mebutate gel, 0.015%, in patients with actinic keratosis

    PubMed Central

    Goldenberg, Gary; Berman, Brian

    2015-01-01

    Lesion-directed and field-directed therapies are used to treat actinic keratosis (AK). Therapeutic approaches that combine both types of therapies may improve the successful elimination of AKs. A randomized, double-blind, vehicle-controlled study evaluated the safety, tolerability, and efficacy of topical field treatment with ingenol mebutate gel, 0.015%, after cryosurgery to AKs on the face and scalp. Patients with 4–8 visible discrete AKs in a 25-cm2 contiguous area received cryosurgery of all AKs at baseline. After a 3-week healing period, patients applied ingenol mebutate gel, 0.015%, or vehicle gel once daily for 3 consecutive days to the treatment area. The incidence, severity, and time course of the development and resolution of local skin reactions were measured from baseline to week 11. Local skin reactions peaked shortly after completion of ingenol mebutate treatment and generally resolved within 2 weeks. The mean (95% confidence interval) composite score (maximum range, 0–24) for these reactions was higher in patients with treatment of AKs on the face, 9.3 (8.5–10.1), as compared with the scalp, 5.8 (4.3–7.4). Erythema and flaking/scaling were the major contributors to the composite local skin reaction score. These results show that local skin reactions associated with ingenol mebutate treatment of the face or scalp are well tolerated after recent cryosurgery. PMID:25565875

  9. The reconstitution of actin polymerization on liposomes.

    PubMed

    Stamnes, Mark; Xu, Weidong

    2010-01-01

    Membrane-associated actin polymerization is of considerable interest due to its role in cell migration and the motility of intracellular organelles. Intensive research efforts are underway to investigate the physiological role of membrane-associated actin as well as the regulation and mechanics of actin assembly. Branched actin polymerization on membranes is catalyzed by the Arp2/3 complex. Signaling events leading to the activation of the guanosine triphosphate (GTP)-binding protein Cdc42 stimulate Arp2/3-dependent actin polymerization. We have studied the role of Cdc42 at the Golgi apparatus in part by reconstituting actin polymerization on isolated Golgi membranes and on liposomes. In this manner, we showed that cytosolic proteins are sufficient for actin assembly on a phospholipid bilayer. Here we describe methods for the cell-free reconstitution of membrane-associated actin polymerization using liposomes and brain cytosol.

  10. Dynamic actin structures stabilized by profilin.

    PubMed Central

    Finkel, T; Theriot, J A; Dise, K R; Tomaselli, G F; Goldschmidt-Clermont, P J

    1994-01-01

    We describe the production and analysis of clonal cell lines in which we have overexpressed human profilin, a small ubiquitous actin monomer binding protein, to assess the role of profilin on actin function in vivo. The concentration of filamentous actin is increased in cells with higher profilin levels, and actin filament half-life measured in these cells is directly proportional to the steady-state profilin concentration. The distribution of actin filaments is altered by profilin overexpression. While parallel actin bundles crossing the cells are virtually absent in cells overexpressing profilin, the submembranous actin network of these cells is denser than in control cells. These results suggest that in vivo profilin regulates the stability, and thereby distribution, of specific dynamic actin structures. Images PMID:8108438

  11. Association of actin with alpha crystallins

    NASA Technical Reports Server (NTRS)

    Gopalakrishnan, S.; Boyle, D.; Takemoto, L.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    The alpha crystallins are cytosolic proteins that co-localize and co-purify with actin-containing microfilaments. Affinity column chromatography employing both covalently-coupled actin or alpha crystallin was used to demonstrate specific and saturable binding of actin with alpha crystallin. This conclusion was confirmed by direct visualization of alpha aggregates bound to actin polymerized in vitro. The significance of this interaction in relation to the functional properties of these two polypeptides will be discussed.

  12. Actin in hair cells and hearing loss.

    PubMed

    Drummond, Meghan C; Belyantseva, Inna A; Friderici, Karen H; Friedman, Thomas B

    2012-06-01

    Hereditary deafness is genetically heterogeneous such that mutations of many different genes can cause hearing loss. This review focuses on the evidence and implications that several of these deafness genes encode actin-interacting proteins or actin itself. There is a growing appreciation of the contribution of the actin interactome in stereocilia development, maintenance, mechanotransduction and malfunction of the auditory system.

  13. Yersinia effector YopO uses actin as bait to phosphorylate proteins that regulate actin polymerization

    PubMed Central

    Lee, Wei Lin; Grimes, Jonathan M; Robinson, Robert C

    2016-01-01

    Pathogenic Yersinia species evade host immune systems through the injection of Yersinia outer proteins (Yops) into phagocytic cells. One Yop, YopO, also known as YpkA, induces actin-filament disruption, impairing phagocytosis. Here we describe the X-ray structure of Yersinia enterocolitica YopO in complex with actin, which reveals that YopO binds to an actin monomer in a manner that blocks polymerization yet allows the bound actin to interact with host actin-regulating proteins. SILAC-MS and biochemical analyses confirm that actin-polymerization regulators such as VASP, EVL, WASP, gelsolin and the formin diaphanous 1 are directly sequestered and phosphorylated by YopO through formation of ternary complexes with actin. This leads to a model in which YopO at the membrane sequesters actin from polymerization while using the bound actin as bait to recruit, phosphorylate and misregulate host actin-regulating proteins to disrupt phagocytosis. PMID:25664724

  14. Yersinia effector YopO uses actin as bait to phosphorylate proteins that regulate actin polymerization.

    PubMed

    Lee, Wei Lin; Grimes, Jonathan M; Robinson, Robert C

    2015-03-01

    Pathogenic Yersinia species evade host immune systems through the injection of Yersinia outer proteins (Yops) into phagocytic cells. One Yop, YopO, also known as YpkA, induces actin-filament disruption, impairing phagocytosis. Here we describe the X-ray structure of Yersinia enterocolitica YopO in complex with actin, which reveals that YopO binds to an actin monomer in a manner that blocks polymerization yet allows the bound actin to interact with host actin-regulating proteins. SILAC-MS and biochemical analyses confirm that actin-polymerization regulators such as VASP, EVL, WASP, gelsolin and the formin diaphanous 1 are directly sequestered and phosphorylated by YopO through formation of ternary complexes with actin. This leads to a model in which YopO at the membrane sequesters actin from polymerization while using the bound actin as bait to recruit, phosphorylate and misregulate host actin-regulating proteins to disrupt phagocytosis.

  15. What is the role of field-directed therapy in the treatment of actinic keratosis? Part 1: overview and investigational topical agents.

    PubMed

    Berman, Brian; Cohen, David E; Amini, Sadegh

    2012-05-01

    Actinic keratosis (AK) constitutes the initial epidermal lesion in a disease continuum that may progress to invasive squamous cell carcinoma (SCC). A number of treatment options are available to clear lesions, and thus reduce the risk for progression. Field-directed approaches are primarily used to clear multiple AKs and subclinical lesions. Current field-directed approaches still have a number of unmet needs, and a number of investigational agents are being evaluated. Topical therapy can be improved by shortening treatment periods; enhancing tolerability, compliance, and patient satisfaction; reducing recurrence rates; and lowering cost. This 2-part review will explain the role of field-directed therapy in the treatment of AK. Part 1 focuses mainly on investigational agents that are being studied for topical patient-administered, field-directed therapy. PMID:22768439

  16. Mechanism of Actin-Based Motility

    NASA Astrophysics Data System (ADS)

    Pantaloni, Dominique; Le Clainche, Christophe; Carlier, Marie-France

    2001-05-01

    Spatially controlled polymerization of actin is at the origin of cell motility and is responsible for the formation of cellular protrusions like lamellipodia. The pathogens Listeria monocytogenes and Shigella flexneri, which undergo actin-based propulsion, are acknowledged models of the leading edge of lamellipodia. Actin-based motility of the bacteria or of functionalized microspheres can be reconstituted in vitro from only five pure proteins. Movement results from the regulated site-directed treadmilling of actin filaments, consistent with observations of actin dynamics in living motile cells and with the biochemical properties of the components of the synthetic motility medium.

  17. 46 CFR 7.150 - Canadian (BC) and United States (AK) Borders to Cape Spencer, AK.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 1 2014-10-01 2014-10-01 false Canadian (BC) and United States (AK) Borders to Cape Spencer, AK. 7.150 Section 7.150 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY PROCEDURES APPLICABLE TO THE PUBLIC BOUNDARY LINES Alaska § 7.150 Canadian (BC) and United States (AK) Borders to...

  18. 46 CFR 7.150 - Canadian (BC) and United States (AK) Borders to Cape Spencer, AK.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 1 2011-10-01 2011-10-01 false Canadian (BC) and United States (AK) Borders to Cape Spencer, AK. 7.150 Section 7.150 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY PROCEDURES APPLICABLE TO THE PUBLIC BOUNDARY LINES Alaska § 7.150 Canadian (BC) and United States (AK) Borders to...

  19. 46 CFR 7.150 - Canadian (BC) and United States (AK) Borders to Cape Spencer, AK.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 1 2012-10-01 2012-10-01 false Canadian (BC) and United States (AK) Borders to Cape Spencer, AK. 7.150 Section 7.150 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY PROCEDURES APPLICABLE TO THE PUBLIC BOUNDARY LINES Alaska § 7.150 Canadian (BC) and United States (AK) Borders to...

  20. 46 CFR 7.150 - Canadian (BC) and United States (AK) Borders to Cape Spencer, AK.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 1 2013-10-01 2013-10-01 false Canadian (BC) and United States (AK) Borders to Cape Spencer, AK. 7.150 Section 7.150 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY PROCEDURES APPLICABLE TO THE PUBLIC BOUNDARY LINES Alaska § 7.150 Canadian (BC) and United States (AK) Borders to...

  1. 46 CFR 7.150 - Canadian (BC) and United States (AK) Borders to Cape Spencer, AK.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 1 2010-10-01 2010-10-01 false Canadian (BC) and United States (AK) Borders to Cape Spencer, AK. 7.150 Section 7.150 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY PROCEDURES APPLICABLE TO THE PUBLIC BOUNDARY LINES Alaska § 7.150 Canadian (BC) and United States (AK) Borders to...

  2. The actin of muscle and fibroblasts.

    PubMed Central

    Anderson, P J

    1976-01-01

    The isolation and quantification of an 18-residue peptide from the N-terminal region of chicken actin was used to quantify the amount of actin in acetone-dried powders of chicken breast muscle and chicken-embryo fibroblasts. Either isotope dilution or double labelling can be used for peptide quantification. About 17% of the protein of chicken breast muscle was estimated to be actin. However, only 0.25% of the protein of chicken-embryo fibroblasts was determined to be actin by quantification of this peptide. The actin content of fibroblasts may be low or the amino acid sequences of muscle and fibroblast actin may differ in the N-terminal region. The methodology used can be extended to examine whether other regions of muscle actin sequence are present in fibroblasts or other cell types. PMID:938480

  3. Quantifying actin wave modulation on periodic topography

    NASA Astrophysics Data System (ADS)

    Guven, Can; Driscoll, Meghan; Sun, Xiaoyu; Parker, Joshua; Fourkas, John; Carlsson, Anders; Losert, Wolfgang

    2014-03-01

    Actin is the essential builder of the cell cytoskeleton, whose dynamics are responsible for generating the necessary forces for the formation of protrusions. By exposing amoeboid cells to periodic topographical cues, we show that actin can be directionally guided via inducing preferential polymerization waves. To quantify the dynamics of these actin waves and their interaction with the substrate, we modify a technique from computer vision called ``optical flow.'' We obtain vectors that represent the apparent actin flow and cluster these vectors to obtain patches of newly polymerized actin, which represent actin waves. Using this technique, we compare experimental results, including speed distribution of waves and distance from the wave centroid to the closest ridge, with actin polymerization simulations. We hypothesize the modulation of the activity of nucleation promotion factors on ridges (elevated regions of the surface) as a potential mechanism for the wave-substrate coupling. Funded by NIH grant R01GM085574.

  4. Photodynamic Therapy with Ablative Carbon Dioxide Fractional Laser in Treatment of Actinic Keratosis

    PubMed Central

    Jang, Yong Hyun; Lee, Dong Jun; Shin, Jaeyoung; Kang, Hee Young; Lee, Eun-So

    2013-01-01

    Background Recently, photodynamic therapy (PDT) has been shown to be an effective first-line treatment for actinic keratosis (AK). However, a major limitation of PDT is the long incubation time required to allow penetration of the photosensitizer. Objective The aim of this study was to assess if pretreatment with an ablative carbon dioxide (CO2) fractional laser can reduce the incubation time of the photosensitizer. Methods Initially, 29 patients with a total of 34 AK lesions were treated with an ablative CO2 fractional laser at Ajou University Hospital between January and December 2010. Immediately after the laser treatment, topical 20% 5-aminolevulinic acid or methyl-aminolevulinate was applied to the AK lesions and incubated for 70 to 90 minutes. Then, the treated areas were illuminated with a red light source. Improvement was clinically or histologically assessed eight weeks after the treatment. Results In spite of the short incubation time, 24 lesions (70.6%) showed a complete response (CR) within three sessions of PDT (10 lesions a clinical CR and 14 lesions a clinical/histological CR). There were no significant side effects associated with the combination of ablative CO2 fractional laser and PDT. Conclusion Ablative CO2 fractional laser may be considered an additional treatment option for reducing the incubation time of the photosensitizer in PDT. PMID:24371387

  5. Laser-mediated Photodynamic Therapy: An Alternative Treatment for Actinic Keratosis?

    PubMed

    Kessels, Janneke P H M; Nelemans, Patty J; Mosterd, Klara; Kelleners-Smeets, Nicole W J; Krekels, Gertruud A M; Ostertag, Judith U

    2016-03-01

    Photodynamic therapy (PDT) with light emitting diode (LED) illumination is a frequently used treatment modality for actinic keratosis (AK) with excellent cosmetic outcome. A major disadvantage, however, is the high pain score. Pulsed dye laser (PDL) illumination has been suggested, but the long-term efficacy of this treatment is unknown. In this split-face study we prospectively treated 61 patients with AK, with both LED-PDT and PDL-PDT. The mean change in the number of lesions between the end of follow-up and start of therapy was -4.25 (95% confidence interval (95% CI) -5.07; -3.43) for LED-PDT and -3.88 (95% CI -4,76; -2.99) for PDL-PDT, with a non-significant difference (p = 0.258) of -0.46 (95% CI -1.28; 0.35). The percentage decrease from baseline in the total number of AK was 55.8% and 47.8%, respectively, at 12-month follow-up. Visual analogue scale pain score was lower after PDL (mean 2.64) compared with LED illumination (mean 6.47). These findings indicate that PDL-PDT is an effective alternative illumination source fo. PMID:26551377

  6. Evidence for a species of nuclear actin distinct from cytoplasmic and muscles actins.

    PubMed

    Bremer, J W; Busch, H; Yeoman, L C

    1981-03-31

    Nuclear actin (protein BJ) has been isolated from the chromatin of Novikoff hepatoma ascites cells and purified to homogeneity by selective extraction, Sepharose CL-6B chromatography, and preparative polyacrylamide gel electrophoresis. A comparison of nuclear and cytoplasmic actins from Novikoff hepatoma cells and rabbit muscle actin was made by amino acid analysis, isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and two-dimensional peptide mapping procedures. By these criteria, all of the proteins compared are actins, but each is chemically distinct. It was concluded, therefore, that nuclear actin is similar to, but not identical with, cytoplasmic actin isolated from Novikoff hepatoma cells. A striking similarity in peptide charge and migration as shown by peptide map analysis was observed for nuclear and rabbit skeletal muscle actins. This may indicate that nuclear actin has the capacity for contractile function. In addition, the actins synthesized in Novikoff hepatoma cells may results from more than two structural genes.

  7. Affinity chromatography of immobilized actin and myosin.

    PubMed Central

    Bottomley, R C; Trayer, I P

    1975-01-01

    Actin and myosin were immobilized by coupling them to agarose matrices. Both immobilized G-actin and immobilized myosin retain most of the properties of the proteins in free solution and are reliable over long periods of time. Sepharose-F-actin, under the conditions used in this study, has proved unstable and variable in its properties. Sepharose-G-actin columns were used to bind heavy meromyosin and myosin subfragment 1 specifically and reversibly. The interaction involved is sensitive to variation in ionic strength, such that myosin itself is not retained by the columns at the high salt concentration required for its complete solubilization. Myosin, rendered soluble at low ionic strength by polyalanylation, will interact successfully with the immobilized actin. The latter can distinguish between active and inactive fractions of the proteolytic and polyalanyl myosin derivatives, and was used in the preparation of these molecules. The complexes formed between the myosin derivatives and Sepharose-G-actin can be dissociated by low concentrations of ATP, ADP and pyrophosphate in both the presence and the absence of Mg2+. The G-actin columns were used to evaluate the results of chemical modifications of myosin subfragments on their interactions with actin. F-Actin in free solution is bound specifically and reversibly to columns of insolubilized myosin. Thus, with elution by either ATP or pyrophosphate, actin has been purified in one step from extracts of acetone-dried muscle powder. PMID:241335

  8. Multiple actin binding domains of Ena/VASP proteins determine actin network stiffening.

    PubMed

    Gentry, Brian S; van der Meulen, Stef; Noguera, Philippe; Alonso-Latorre, Baldomero; Plastino, Julie; Koenderink, Gijsje H

    2012-11-01

    Vasodilator-stimulated phosphoprotein (Ena/VASP) is an actin binding protein, important for actin dynamics in motile cells and developing organisms. Though VASP's main activity is the promotion of barbed end growth, it has an F-actin binding site and can form tetramers, and so could additionally play a role in actin crosslinking and bundling in the cell. To test this activity, we performed rheology of reconstituted actin networks in the presence of wild-type VASP or mutants lacking the ability to tetramerize or to bind G-actin and/or F-actin. We show that increasing amounts of wild-type VASP increase network stiffness up to a certain point, beyond which stiffness actually decreases with increasing VASP concentration. The maximum stiffness is 10-fold higher than for pure actin networks. Confocal microscopy shows that VASP forms clustered actin filament bundles, explaining the reduction in network elasticity at high VASP concentration. Removal of the tetramerization site results in significantly reduced bundling and bundle clustering, indicating that VASP's flexible tetrameric structure causes clustering. Removing either the F-actin or the G-actin binding site diminishes VASP's effect on elasticity, but does not eliminate it. Mutating the F-actin and G-actin binding site together, or mutating the F-actin binding site and saturating the G-actin binding site with monomeric actin, eliminates VASP's ability to increase network stiffness. We propose that, in the cell, VASP crosslinking confers only moderate increases in linear network elasticity, and unlike other crosslinkers, VASP's network stiffening activity may be tuned by the local concentration of monomeric actin.

  9. The actin cytoskeleton in endothelial cell phenotypes

    PubMed Central

    Prasain, Nutan; Stevens, Troy

    2009-01-01

    Endothelium forms a semi-permeable barrier that separates blood from the underlying tissue. Barrier function is largely determined by cell-cell and cell-matrix adhesions that define the limits of cell borders. Yet, such cell-cell and cell-matrix tethering is critically reliant upon the nature of adherence within the cell itself. Indeed, the actin cytoskeleton fulfills this essential function, to provide a strong, dynamic intracellular scaffold that organizes integral membrane proteins with the cell’s interior, and responds to environmental cues to orchestrate appropriate cell shape. The actin cytoskeleton is comprised of three distinct, but interrelated structures, including actin cross-linking of spectrin within the membrane skeleton, the cortical actin rim, and actomyosin-based stress fibers. This review addresses each of these actin-based structures, and discusses cellular signals that control the disposition of actin in different endothelial cell phenotypes. PMID:19028505

  10. Efficacy of Photodynamic Therapy in the Short and Medium Term in the Treatment of Actinic Keratosis, Basal Cell Carcinoma, Acne Vulgaris and Photoaging: Results from Four Clinical Trials

    PubMed Central

    Martínez-Carpio, PA; Alcolea-López, JM; Vélez, M

    2012-01-01

    Objective: To determine the clinical efficacy of methyl-aminolevulinate (MAL)-Photodynamic Therapy (PDT) in the treatment of actinic keratosis (AK), basal cell carcinoma (BCC), acne vulgaris (AV) and photoaging (PA), in the short and medium term. Subjects and methods: Four separate prospective studies were designed on patients with AK (n=25), BCC (n=20), AV (n=20) and PA (n=25). Two PDT protocols were applied, and different clinical efficacy criteria were established, including lesion count and size. Two semi-quantitative and four analogue visual scales were completed for the evaluation of results according to the therapist, the patient and two independent experts. Results: In the AK and BCC studies, full clinical remission was observed in 84.7% and 75.7% of lesions, respectively. In the AV study, the number of inflammatory and non-inflammatory lesions fell significantly (p<0.001, p<0.05). In the PA study a reduction in Dover scale scores (3.19 vs. 2.14, p<0.001) was proven. The percentages of satisfied or very satisfied patients were: AK=88%, BCC=90%, AV=89% and PA=80%. A year later, none of the AK or BCC lesions had reappeared, and the cases of AV and PA remained stable, with a tendency towards improvement. Conclusion: the MAL-PDT procedures used produced efficacious, safe and satisfactory results in KA, BCC, AV and PA in the short and medium term. PMID:24511190

  11. Filopodia-like actin cables position nuclei in association with perinuclear actin in Drosophila nurse cells.

    PubMed

    Huelsmann, Sven; Ylänne, Jari; Brown, Nicholas H

    2013-09-30

    Controlling the position of the nucleus is vital for a number of cellular processes from yeast to humans. In Drosophila nurse cells, nuclear positioning is crucial during dumping, when nurse cells contract and expel their contents into the oocyte. We provide evidence that in nurse cells, continuous filopodia-like actin cables, growing from the plasma membrane and extending to the nucleus, achieve nuclear positioning. These actin cables move nuclei away from ring canals. When nurse cells contract, actin cables associate laterally with the nuclei, in some cases inducing nuclear turning so that actin cables become partially wound around the nuclei. Our data suggest that a perinuclear actin meshwork connects actin cables to nuclei via actin-crosslinking proteins such as the filamin Cheerio. We provide a revised model for how actin structures position nuclei in nurse cells, employing evolutionary conserved machinery.

  12. Persistent nuclear actin filaments inhibit transcription by RNA polymerase II.

    PubMed

    Serebryannyy, Leonid A; Parilla, Megan; Annibale, Paolo; Cruz, Christina M; Laster, Kyle; Gratton, Enrico; Kudryashov, Dmitri; Kosak, Steven T; Gottardi, Cara J; de Lanerolle, Primal

    2016-09-15

    Actin is abundant in the nucleus and it is clear that nuclear actin has important functions. However, mystery surrounds the absence of classical actin filaments in the nucleus. To address this question, we investigated how polymerizing nuclear actin into persistent nuclear actin filaments affected transcription by RNA polymerase II. Nuclear filaments impaired nuclear actin dynamics by polymerizing and sequestering nuclear actin. Polymerizing actin into stable nuclear filaments disrupted the interaction of actin with RNA polymerase II and correlated with impaired RNA polymerase II localization, dynamics, gene recruitment, and reduced global transcription and cell proliferation. Polymerizing and crosslinking nuclear actin in vitro similarly disrupted the actin-RNA-polymerase-II interaction and inhibited transcription. These data rationalize the general absence of stable actin filaments in mammalian somatic nuclei. They also suggest a dynamic pool of nuclear actin is required for the proper localization and activity of RNA polymerase II.

  13. Mycosis fungoides patient accompanied actinic keratosis, actinic keratosis with squamous cell carcinoma transformation, and porokeratosis after NBUVB therapy – 1st case report and review of the literature

    PubMed Central

    Zhao, Meng-jie; Abdul-fattah, Bilal; Qu, Xiao-ying; Wang, Cui-yan; Wang, Xia; Ran, Yi; Lai, Ting; Chen, Si-yuan; Huang, Chang-zheng

    2016-01-01

    Abstract Introduction: Mycosis fungoides (MF) is the most common form of primary cutaneous T cell lymphoma. Narrowband ultraviolet B light (NBUVB) is used increasingly in treating MF because of its good toleration and well-established management. Concerns: To discuss the risk factors and underlying pathogenic factors in the patients with secondary skin diseases after NBUVB therapy. Methods: We report in details the first case of a patient with MF accompanied with actinic keratosis (AK), AK with squamous cell carcinoma (SCC) transformation and porokeratosis after NBUVB therapy. Meanwhile, Sequence variants in tumor suppressor p53 gene in the patient's specimens were detected. A literature search of the key word “narrowband ultraviolet B light ”and “side effects” was performed on PubMed, 14 cases of this entity were found. A total of 15 patients including our case were reviewed in this study and meaningful conclusion could be drawn. Outcomes: The mean age at diagnosis of secondary skin dermatoses after NBUVB therapy was 62.08 years with a male to female ratio of 2:1. The cases were reported more in Europeans than in Asians (2.75:1), and the Fitzpatrick skin type was mainly Ito III (12/15). The mean cumulative number and cumulative dose of UVB treatments were 43.71 and 42, 400 (mJ/cm2), respectively. There was a positive relationship between Fitzpatrick skin type and cumulative dose of UVB treatments. Among the secondary skin diseases after NBUVB treatment, 12 were tumors, 2 were non-tumorous dermatoses. Only our patient presented with both. By polymerase chain reaction-single nucleotide polymorphism (PCR-SNP) analysis, C–G mutation of exon 4 of p53 was found in AK and MF specimens in our patient. Conclusion: To our knowledge, our case is the first MF patient accompanied with AK, AK with SCC transformation and Porokeratosis after NBUVB treatment. Lower Fitzpatrick skin type may be the risk factor of secondary skin diseases after NBUVB treatment. PMID

  14. Stochastic model of profilin-actin polymerization

    NASA Astrophysics Data System (ADS)

    Horan, Brandon; Vavylonis, Dimitrios

    A driving factor in cell motility and other processes that involve changes of cell shape is the rapid polymerization of actin subunits into long filaments. This process is regulated by profilin, a protein which binds to actin subunits and regulates elongation of actin filaments. Whether profilin stimulates polymerization by coupling to hydrolysis of ATP-bound actin is debated. Previous studies have proposed indirect coupling to ATP hydrolysis using rate equations, but did not include the effects of fluctuations that are important near the critical concentration. We developed stochastic simulations using the Gillespie algorithm to study single filament elongation at the barbed end in the presence of profilin. We used recently measured rate constants and estimated the rate of profilin binding to the barbed end such that detailed balance is satisfied. Fast phosphate release at the tip of the filament was accounted for. The elongation rate and length diffusivity as functions of profilin and actin concentration were calculated and used to extract the critical concentrations of free actin and of total actin. We show under what conditions profilin leads to an increase in the critical concentration of total actin but a decrease in the critical concentration of free actin.

  15. Contribution of nuclear actin to transcription regulation.

    PubMed

    Yamazaki, Shota; Yamamoto, Koji; Harata, Masahiko

    2015-06-01

    Actin, an integral component of the cytoskeleton, plays crucial roles in a variety of cell functions, including cell migration, adhesion, polarity and shape change. Studies performed during the last couple of decades have revealed that the actin also exists in the nucleus. However, the function and properties of nuclear actin remained elusive so far. Recently, we showed that an actin tagged with EYFP and fused with a nuclear localization signal (EYFP-NLS-actin) formed visible filamentous (F)-actin bundles in cells. To obtain further details about the individual genes that are affected by the nuclear actin, we have used the microarray analysis to determine the changes in the expression levels of RNAs in HeLa cells as a result of EYFP-NLS-actin expression. Our results suggest that the nuclear actin plays a role in the activation of genes rather than their repression. The data has been deposited in the Gene Expression Omnibus (GEO) database under the accession number GSE59799.

  16. Phosphorylation and actin activation of brain myosin.

    PubMed Central

    Barylko, B; Sobieszek, A

    1983-01-01

    A method is described for obtaining brain myosin that shows significant actin activation, after phosphorylation with chicken gizzard myosin light chain kinase. Myosin with this activity could be obtained only via the initial purification of brain actomyosin. The latter complex, isolated by a method similar to that used for smooth muscle, contained actin, myosin, tropomyosin of the non-muscle type and another actin-binding protein of approximately 100,000 daltons. From the presence of a specific myosin light chain kinase and phosphatase in brain tissue it is suggested that the regulation of actin-myosin interaction operates via phosphorylation and dephosphorylation of myosin. Images Fig. 1. Fig. 3. PMID:11894951

  17. Architecture and Connectivity Govern Actin Network Contractility.

    PubMed

    Ennomani, Hajer; Letort, Gaëlle; Guérin, Christophe; Martiel, Jean-Louis; Cao, Wenxiang; Nédélec, François; De La Cruz, Enrique M; Théry, Manuel; Blanchoin, Laurent

    2016-03-01

    Actomyosin contractility plays a central role in a wide range of cellular processes, including the establishment of cell polarity, cell migration, tissue integrity, and morphogenesis during development. The contractile response is variable and depends on actomyosin network architecture and biochemical composition. To determine how this coupling regulates actomyosin-driven contraction, we used a micropatterning method that enables the spatial control of actin assembly. We generated a variety of actin templates and measured how defined actin structures respond to myosin-induced forces. We found that the same actin filament crosslinkers either enhance or inhibit the contractility of a network, depending on the organization of actin within the network. Numerical simulations unified the roles of actin filament branching and crosslinking during actomyosin contraction. Specifically, we introduce the concept of "network connectivity" and show that the contractions of distinct actin architectures are described by the same master curve when considering their degree of connectivity. This makes it possible to predict the dynamic response of defined actin structures to transient changes in connectivity. We propose that, depending on the connectivity and the architecture, network contraction is dominated by either sarcomeric-like or buckling mechanisms. More generally, this study reveals how actin network contractility depends on its architecture under a defined set of biochemical conditions.

  18. Real-life practice study of the clinical outcome and cost-effectiveness of photodynamic therapy using methyl aminolevulinate (MAL-PDT) in the management of actinic keratosis and basal cell carcinoma.

    PubMed

    Annemans, Lieven; Caekelbergh, Karin; Roelandts, Rik; Boonen, Hugo; Leys, Christoph; Nikkels, Arjen F; van Den Haute, V; van Quickenborne, L; Verhaeghe, Evelien; Leroy, Bernard

    2008-01-01

    Clinical trials have shown that photodynamic therapy using methyl aminolevulinate (MAL-PDT) is an effective treatment for actinic keratosis (AK), and nodular and superficial basal cell carcinoma (nBCC and sBCC) unsuitable for other available therapies. Economic evaluation models have shown that it is a cost effective intervention as well. The objectives of this prospective, observational, one arm study were (i) to verify in a real-life practice study the results obtained in previous clinical trials with MAL-PDT in the treatment of AK, nBCC and sBCC; (ii) to calculate the real-life cost of treatment and validate predictions from an economic evaluation model. Patients with AK and/or BCC were selected according to Belgian reimbursement criteria for treatment with MAL-PDT. Clinical response, cosmetic outcome and tolerability were assessed. MAL-PDT cost was calculated and compared to published model cost data. Data were collected from 247 patients (117 AK, 130 BCC). A complete clinical response was obtained for 83% of AK (85/102) and BCC (97/116) patients. A good or excellent cosmetic outcome was obtained for 95% of AK patients and 93% of BCC patients. Tolerability was good: only 2 patients withdrew for adverse events. Clinical results were similar to previous studies. Total cost of care per patient was euro 381 for AK, euro 318 for nBCC, and euro 298 for sBCC. Total cost per lesion was euro 58 for AK (identical to model prediction), euro 316 for nBCC and euro 178 for sBCC (both within 20% of model prediction). The clinical results of MAL-PDT in this real-life practice study confirm those demonstrated in previous clinical trials. Costs calculated from this study confirm predicted cost-effectiveness in the original model for MAL-PDT in the management of AK and BCC.

  19. F-actin waves, actin cortex disassembly and focal exocytosis driven by actin-phosphoinositide positive feedback.

    PubMed

    Masters, Thomas A; Sheetz, Michael P; Gauthier, Nils C

    2016-04-01

    Actin polymerization is controlled by the phosphoinositide composition of the plasma membrane. However, the molecular mechanisms underlying the spatiotemporal regulation of actin network organization over extended length scales are still unclear. To observe phosphoinositide-dependent cytoskeletal dynamics we combined the model system of frustrated phagocytosis, total internal reflection microscopy and manipulation of the buffer tonicity. We found that macrophages interacting with IgG-coated glass substrates formed circular F-actin waves on their ventral surface enclosing a region of plasma membrane devoid of cortical actin. Plasma membrane free of actin cortex was strongly depleted of PI(4,5)P2 , but enriched in PI(3,4)P2 and displayed a fivefold increase in exocytosis. Wave formation could be promoted by application of a hypotonic shock. The actin waves were characteristic of a bistable wavefront at the boundary between the regions of membrane containing and lacking cortical actin. Phosphoinositide modifiers and RhoGTPase activities dramatically redistributed with respect to the wavefronts, which often exhibited spatial oscillations. Perturbation of either lipid or actin cytoskeleton-related pathways led to rapid loss of both the polarized lipid distribution and the wavefront. As waves travelled over the plasma membrane, wavefront actin was seen to rapidly polymerize and depolymerize at pre-existing clusters of FcγRIIA, coincident with rapid changes in lipid composition. Thus the potential of receptors to support rapid F-actin polymerization appears to depend acutely on the local concentrations of multiple lipid species. We propose that interdependence through positive feedback from the cytoskeleton to lipid modifiers leads to coordinated local cortex remodeling, focal exocytosis, and organizes extended actin networks. PMID:26915738

  20. F-actin waves, actin cortex disassembly and focal exocytosis driven by actin-phosphoinositide positive feedback.

    PubMed

    Masters, Thomas A; Sheetz, Michael P; Gauthier, Nils C

    2016-04-01

    Actin polymerization is controlled by the phosphoinositide composition of the plasma membrane. However, the molecular mechanisms underlying the spatiotemporal regulation of actin network organization over extended length scales are still unclear. To observe phosphoinositide-dependent cytoskeletal dynamics we combined the model system of frustrated phagocytosis, total internal reflection microscopy and manipulation of the buffer tonicity. We found that macrophages interacting with IgG-coated glass substrates formed circular F-actin waves on their ventral surface enclosing a region of plasma membrane devoid of cortical actin. Plasma membrane free of actin cortex was strongly depleted of PI(4,5)P2 , but enriched in PI(3,4)P2 and displayed a fivefold increase in exocytosis. Wave formation could be promoted by application of a hypotonic shock. The actin waves were characteristic of a bistable wavefront at the boundary between the regions of membrane containing and lacking cortical actin. Phosphoinositide modifiers and RhoGTPase activities dramatically redistributed with respect to the wavefronts, which often exhibited spatial oscillations. Perturbation of either lipid or actin cytoskeleton-related pathways led to rapid loss of both the polarized lipid distribution and the wavefront. As waves travelled over the plasma membrane, wavefront actin was seen to rapidly polymerize and depolymerize at pre-existing clusters of FcγRIIA, coincident with rapid changes in lipid composition. Thus the potential of receptors to support rapid F-actin polymerization appears to depend acutely on the local concentrations of multiple lipid species. We propose that interdependence through positive feedback from the cytoskeleton to lipid modifiers leads to coordinated local cortex remodeling, focal exocytosis, and organizes extended actin networks.

  1. The pros and cons of common actin labeling tools for visualizing actin dynamics during Drosophila oogenesis

    PubMed Central

    Spracklen, Andrew J.; Fagan, Tiffany N.; Lovander, Kaylee E.; Tootle, Tina L.

    2015-01-01

    Dynamic remodeling of the actin cytoskeleton is required for both development and tissue homeostasis. While fixed image analysis has provided significant insight into such events, a complete understanding of cytoskeletal dynamics requires live imaging. Numerous tools for the live imaging of actin have been generated by fusing the actin-binding domain from an actin-interacting protein to a fluorescent protein. Here we comparatively assess the utility of three such tools – Utrophin, Lifeact, and F-tractin – for characterizing the actin remodeling events occurring within the germline-derived nurse cells during Drosophila mid-oogenesis or follicle development. Specifically, we used the UAS/GAL4 system to express these tools at different levels and in different cells, and analyzed these tools for effects on fertility, alterations in the actin cytoskeleton, and ability to label filamentous actin (F-actin) structures by both fixed and live imaging. While both Utrophin and Lifeact robustly label F-actin structures within the Drosophila germline, when strongly expressed they cause sterility and severe actin defects including cortical actin breakdown resulting in multi-nucleate nurse cells, early F-actin filament and aggregate formation during stage 9 (S9), and disorganized parallel actin filament bundles during stage 10B (S10B). However, by using a weaker germline GAL4 driver in combination with a higher temperature, Utrophin can label F-actin with minimal defects. Additionally, strong Utrophin expression within the germline causes F-actin formation in the nurse cell nuclei and germinal vesicle during mid-oogenesis. Similarly, Lifeact expression results in nuclear F-actin only within the germinal vesicle. F-tractin expresses at a lower level than the other two labeling tools, but labels cytoplasmic F-actin structures well without causing sterility or striking actin defects. Together these studies reveal how critical it is to evaluate the utility of each actin labeling

  2. The pros and cons of common actin labeling tools for visualizing actin dynamics during Drosophila oogenesis.

    PubMed

    Spracklen, Andrew J; Fagan, Tiffany N; Lovander, Kaylee E; Tootle, Tina L

    2014-09-15

    Dynamic remodeling of the actin cytoskeleton is required for both development and tissue homeostasis. While fixed image analysis has provided significant insight into such events, a complete understanding of cytoskeletal dynamics requires live imaging. Numerous tools for the live imaging of actin have been generated by fusing the actin-binding domain from an actin-interacting protein to a fluorescent protein. Here we comparatively assess the utility of three such tools--Utrophin, Lifeact, and F-tractin--for characterizing the actin remodeling events occurring within the germline-derived nurse cells during Drosophila mid-oogenesis or follicle development. Specifically, we used the UAS/GAL4 system to express these tools at different levels and in different cells, and analyzed these tools for effects on fertility, alterations in the actin cytoskeleton, and ability to label filamentous actin (F-actin) structures by both fixed and live imaging. While both Utrophin and Lifeact robustly label F-actin structures within the Drosophila germline, when strongly expressed they cause sterility and severe actin defects including cortical actin breakdown resulting in multi-nucleate nurse cells, early F-actin filament and aggregate formation during stage 9 (S9), and disorganized parallel actin filament bundles during stage 10B (S10B). However, by using a weaker germline GAL4 driver in combination with a higher temperature, Utrophin can label F-actin with minimal defects. Additionally, strong Utrophin expression within the germline causes F-actin formation in the nurse cell nuclei and germinal vesicle during mid-oogenesis. Similarly, Lifeact expression results in nuclear F-actin only within the germinal vesicle. F-tractin expresses at a lower level than the other two labeling tools, but labels cytoplasmic F-actin structures well without causing sterility or striking actin defects. Together these studies reveal how critical it is to evaluate the utility of each actin labeling tool

  3. Force of an actin spring

    NASA Astrophysics Data System (ADS)

    Shin, Jennifer; Mahadevan, L.; Matsudaira, Paul

    2003-03-01

    The acrosomal process of the horseshoe crab sperm is a novel mechanochemical molecular spring that converts its elastic stain energy to mechanical work upon the chemical activation by Ca2+. Twisted and bent, the initial state of the acrosomal bundle features a high degree of complexity in its structure and the energy is believed to be stored in the highly strained actin filaments as an elastic potential energy. When activated, the bundle relaxes from the coil of the highly twisted and bent filaments to its straight conformation at a mean velocity of 15um/s. The mean extension velocity increases dramatically from 3um/s to 27um/s when temperature of the medium is changed from 9.6C to 32C (respective viscosities of 1.25-0.75cp), yet it exhibits a very weak dependence on changes in the medium viscosity (1cp-33cp). These experiments suggest that the uncoiling of the actin spring should be limited not by the viscosity of the medium but by the unlatching events of involved proteins at a molecular level. Unlike the viscosity-limited processes, where force is directly related to the rate of the reaction, a direct measurement is required to obtain the spring force of the acrosomal process. The extending acrosomal bundle is forced to push against a barrier and its elastic buckling response is analyzed to measure the force generated during the uncoiling.

  4. Effect of alpha-actinin on actin structure. Actin ATPase activity.

    PubMed

    Singh, I; Goll, D E; Robson, R M

    1981-08-28

    Alpha-Actinin increases the ATPase activity of actin by up to 84%, depending un pH, divalent cations present and the added Mg2+: ATP ratio. Dithiothreitol decreases actin ATPase activity approx. 20% but does not reduce the ability of alpha-actinin to increase actin ATP activity. Increasing amounts of added alpha-actinin up to 1 mos alpha-actinin to 49 mol actin cause in increasing increment in actin ATPase activity, but adding alpha-actinin beyond 1 mol alpha-actinin to 49 mol actin elicits only small additional increments in activity. Actin ATPase activity ranges from approx 100 nmol Pi/mg actin per h (4.3 mol Pi/mol actin per h) at high levels (10 mM) of ATP in the presence of lower amounts (1 mM) of added mg2+ to approx. 12.5 nmol Pi/mg actin per h (0.52 mol Pi/mol actin per h) at high pH (8.5) or at low levels (0.5-1.0 mM) of ATP in the presence of higher amounts (10 mM) of added Mg2+ ATp uncomplexed with Mg2+ inhibits the ability of alpha-actinin to increase F-actin ATPase activity. Activities with different divalent cations showed that the actin ATPase in these studies, which was 1/100 as great as Mg2+-modified actomyosin ATPase activity, was not due to trace amounts of myosin contaminating the actin preparations. The results are consistent with the concept that alpha-actinin can alter the structure of actin monomers. PMID:6456018

  5. Change in the actin-myosin subfragment 1 interaction during actin polymerization.

    PubMed

    Chaussepied, P; Kasprzak, A A

    1989-12-01

    To better characterize the conformational differences of G- and F-actin, we have compared the interaction between G- and F-actin with myosin subfragment 1 (S1) which had part of its F-actin binding site (residues 633-642) blocked by a complementary peptide or "antipeptide" (Chaussepied, P., and Morales, M. F. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7471-7475). Light scattering, sedimentation, and electron microscopy measurements showed that, with the antipeptide covalently attached to the S1 heavy chain, S1 was not capable of inducing G-actin polymerization in the absence of salt. Moreover, the antipeptide-carrying S1 did not change the fluorescence polarization of 5-[2-(iodoacetyl)-aminoethyl]aminonaphthalene-1-sulfonic acid (1,5-IAEDANS)-labeled G-actin or of 1,5-IAEDANS-labeled actin dimer, compared to the control S1. This result, interpreted as a lack of interaction between G-actin and antipeptide-carrying S1, was confirmed further by the following experiments: in the presence of G-actin, antipeptide.S1 heavy chain was not protected against trypsin and papain proteolysis, and G-actin could not be cross-linked to antipeptide.S1 by 1-ethyl-3[-3-(dimethylamino)propyl]carbodiimide. In contrast, similar experiments showed that antipeptide.S1 was able to interact with nascent F-actin and with F-actin. Thus, blocking the stretch 633-642 of S1 heavy chain by the antipeptide strongly inhibits G-actin-S1 interaction but only slightly alters F-actin-S1 contact. We, therefore postulate that this stretch of skeletal S1 heavy chain is essential for G-actin-S1 interaction and that the G-F transformation generates new S1 binding site(s) on the actin molecule.

  6. Unconventional actin conformations localize on intermediate filaments in mitosis

    SciTech Connect

    Hubert, Thomas; Vandekerckhove, Joel; Gettemans, Jan

    2011-03-04

    Research highlights: {yields} Unconventional actin conformations colocalize with vimentin on a cage-like structure in metaphase HEK 293T cells. {yields} These conformations are detected with the anti-actin antibodies 1C7 ('lower dimer') and 2G2 ('nuclear actin'), but not C4 (monomeric actin). {yields} Mitotic unconventional actin cables are independent of filamentous actin or microtubules. {yields} Unconventional actin colocalizes with vimentin on a nocodazole-induced perinuclear dense mass of cables. -- Abstract: Different structural conformations of actin have been identified in cells and shown to reside in distinct subcellular locations of cells. In this report, we describe the localization of actin on a cage-like structure in metaphase HEK 293T cells. Actin was detected with the anti-actin antibodies 1C7 and 2G2, but not with the anti-actin antibody C4. Actin contained in this structure is independent of microtubules and actin filaments, and colocalizes with vimentin. Taking advantage of intermediate filament collapse into a perinuclear dense mass of cables when microtubules are depolymerized, we were able to relocalize actin to such structures. We hypothesize that phosphorylation of intermediate filaments at mitosis entry triggers the recruitment of different actin conformations to mitotic intermediate filaments. Storage and partition of the nuclear actin and antiparallel 'lower dimer' actin conformations between daughter cells possibly contribute to gene transcription and transient actin filament dynamics at G1 entry.

  7. Synthetic peptides that cause F-actin bundling and block actin depolymerization

    DOEpatents

    Sederoff, Heike; Huber, Steven C; Larabell, Carolyn A

    2011-10-18

    Synthetic peptides derived from sucrose synthase, and having homology to actin and actin-related proteins, sharing a common motif, useful for causing acting bundling and preventing actin depolymerization. Peptides exhibiting the common motif are described, as well as specific synthetic peptides which caused bundled actin and inhibit actin depolymerization. These peptides can be useful for treating a subject suffering from a disease characterized by cells having neoplastic growth, for anti-cancer therapeutics, delivered to subjects solely, or concomitantly or sequentially with other known cancer therapeutics. These peptides can also be used for stabilizing microfilaments in living cells and inhibiting growth of cells.

  8. Cost-effectiveness analysis of topical treatments for actinic keratosis in the perspective of the Italian health care system.

    PubMed

    Colombo, G L; Chimenti, S; Di Matteo, S; Fargnoli, M C; Frascione, P; Silipo, V; Peris, K

    2010-10-01

    Actinic keratosis (AK) is the most common cutaneous malignant neoplasm and its prevalence continues to increase. According to the most recent findings, AK is currently considered the initial stage, in situ, of squamous cell carcinoma. Field-directed therapies for AKs are the preferred treatment since they have the advantage to clear the clinically visible lesions and also subclinical lesions within the cancerous field. We assessed the cost-effectiveness of topical treatments for AKs including 3% diclofenac in 2.5% hyaluronic acid (HA) gel, imiquimod 5% cream and photodynamic therapy with methyl aminolevulinate (MAL-PDT) in the perspective of the Italian Health Care System (SSN). We used a decision tree analytical approach and efficacy data were drawn from published clinical trials. Cost was evaluated from the SSN perspective during a time horizon of 3 months. A responder was defined as a patient with all lesions clinically cleared and showing an excellent cosmetic result. Based on the applied model, the cost per complete responder was calculated. Diclofenac 3% in HA was less expensive (Euro 256) than MAL-PDT (Euro 320) and imiquimod (Euro 342). Effectiveness was similar and better for diclofenac 3% in HA and MAL-PDT (0.813%) in comparison to 0.734% of imiquimod, respectively. The one-way and probabilistic sensitivity analyses confirmed the results of base case scenario. Based on this cost-effectiveness model, diclofenac 3% in HA can be considered the treatment of choice for AK lesions and surrounding field under a pharmacoeconomic point of view.

  9. Profilin connects actin assembly with microtubule dynamics.

    PubMed

    Nejedla, Michaela; Sadi, Sara; Sulimenko, Vadym; de Almeida, Francisca Nunes; Blom, Hans; Draber, Pavel; Aspenström, Pontus; Karlsson, Roger

    2016-08-01

    Profilin controls actin nucleation and assembly processes in eukaryotic cells. Actin nucleation and elongation promoting factors (NEPFs) such as Ena/VASP, formins, and WASP-family proteins recruit profilin:actin for filament formation. Some of these are found to be microtubule associated, making actin polymerization from microtubule-associated platforms possible. Microtubules are implicated in focal adhesion turnover, cell polarity establishment, and migration, illustrating the coupling between actin and microtubule systems. Here we demonstrate that profilin is functionally linked to microtubules with formins and point to formins as major mediators of this association. To reach this conclusion, we combined different fluorescence microscopy techniques, including superresolution microscopy, with siRNA modulation of profilin expression and drug treatments to interfere with actin dynamics. Our studies show that profilin dynamically associates with microtubules and this fraction of profilin contributes to balance actin assembly during homeostatic cell growth and affects micro-tubule dynamics. Hence profilin functions as a regulator of microtubule (+)-end turnover in addition to being an actin control element.

  10. Probing the actin-auxin oscillator

    PubMed Central

    2010-01-01

    The directional transport of the plant hormone auxin depends on transcellular gradients of auxin-efflux carriers that continuously cycle between plasma membrane and intracellular compartments. This cycling has been proposed to depend on actin filaments. However, the role of actin for the polarity of auxin transport has been disputed. To get insight into this question, actin bundling was induced by overexpression of the actin-binding domain of talin in tobacco BY-2 cells and in rice plants. This bundling can be reverted by addition of auxins, which allows to address the role of actin organization on the flux of auxin. In both systems, the reversion of a normal actin configuration can be restored by addition of exogenous auxins and this fully restores the respective auxin-dependent functions. These findings lead to a model of a self-referring regulatory circuit between polar auxin transport and actin organization. To further dissect the actin-auxin oscillator, we used photoactivated release of caged auxin in tobacco cells to demonstrate that auxin gradients can be manipulated at a subcellular level. PMID:20023411

  11. Profilin connects actin assembly with microtubule dynamics

    PubMed Central

    Nejedla, Michaela; Sadi, Sara; Sulimenko, Vadym; de Almeida, Francisca Nunes; Blom, Hans; Draber, Pavel; Aspenström, Pontus; Karlsson, Roger

    2016-01-01

    Profilin controls actin nucleation and assembly processes in eukaryotic cells. Actin nucleation and elongation promoting factors (NEPFs) such as Ena/VASP, formins, and WASP-family proteins recruit profilin:actin for filament formation. Some of these are found to be microtubule associated, making actin polymerization from microtubule-associated platforms possible. Microtubules are implicated in focal adhesion turnover, cell polarity establishment, and migration, illustrating the coupling between actin and microtubule systems. Here we demonstrate that profilin is functionally linked to microtubules with formins and point to formins as major mediators of this association. To reach this conclusion, we combined different fluorescence microscopy techniques, including superresolution microscopy, with siRNA modulation of profilin expression and drug treatments to interfere with actin dynamics. Our studies show that profilin dynamically associates with microtubules and this fraction of profilin contributes to balance actin assembly during homeostatic cell growth and affects micro­tubule dynamics. Hence profilin functions as a regulator of microtubule (+)-end turnover in addition to being an actin control element. PMID:27307590

  12. Actin cytoskeleton redox proteome oxidation by cadmium

    PubMed Central

    Go, Young-Mi; Orr, Michael

    2013-01-01

    Epidemiological studies associate environmental cadmium (Cd) exposure with the risk of lung diseases. Although mechanisms are not fully elucidated, several studies demonstrate Cd effects on actin and actin-associated proteins. In a recent study of Cd at concentrations similar to environmental exposures, we found that redox-dependent inflammatory signaling by NF-κB was sensitive to the actin-disrupting agent, cytochalasin D. The goal of the present study was to use mass spectrometry-based redox proteomics to investigate Cd effects on the actin cytoskeleton proteome and related functional pathways in lung cells at low environmental concentrations. The results showed that Cd under conditions that did not alter total protein thiols or glutathione redox state caused significant oxidation of peptidyl Cys of proteins regulating actin cytoskeleton. Immunofluorescence microscopy of lung fibroblasts and pulmonary artery endothelial cells showed that low-dose Cd exposure stimulated filamentous actin formation and nuclear localization of destrin, an actin-depolymerizing factor. Taken together, the results show that redox states of peptidyl Cys in proteins associated with actin cytoskeleton pathways are selectively oxidized in lung by Cd at levels thought to occur from environmental exposure. PMID:24077948

  13. Colchicine activates actin polymerization by microtubule depolymerization.

    PubMed

    Jung, H I; Shin, I; Park, Y M; Kang, K W; Ha, K S

    1997-06-30

    Swiss 3T3 fibroblasts were treated with the microtubule-disrupting agent colchicine to study any interaction between microtubule dynamics and actin polymerization. Colchicine increased the amount of filamentous actin (F-actin), in a dose- and time-dependent manner with a significant increase at 1 h by about 130% over control level. Confocal microscopic observation showed that colchicine increased F-actin contents by stress fiber formation without inducing membrane ruffling. Colchicine did not activate phospholipase C and phospholipase D, whereas lysophosphatidic acid did, indicating that colchicine may have a different mechanism of actin polymerization regulation from LPA. A variety of microtubule-disrupting agents stimulated actin polymerization in Swiss 3T3 and Rat-2 fibroblasts as did colchicine, but the microtubule-stabilizing agent taxol inhibited actin polymerization induced by the above microtubule-disrupting agents. In addition, colchicine-induced actin polymerization was blocked by two protein phosphatase inhibitors, okadaic acid and calyculin A. These results suggest that microtubule depolymerization activates stress fiber formation by serine/threonine dephosphorylation in fibroblasts. PMID:9264034

  14. Formin DAAM1 Organizes Actin Filaments in the Cytoplasmic Nodal Actin Network

    PubMed Central

    Luo, Weiwei; Lieu, Zi Zhao; Manser, Ed; Bershadsky, Alexander D.; Sheetz, Michael P.

    2016-01-01

    A nodal cytoplasmic actin network underlies actin cytoplasm cohesion in the absence of stress fibers. We previously described such a network that forms upon Latrunculin A (LatA) treatment, in which formin DAAM1 was localized at these nodes. Knock down of DAAM1 reduced the mobility of actin nodes but the nodes remained. Here we have investigated DAAM1 containing nodes after LatA washout. DAAM1 was found to be distributed between the cytoplasm and the plasma membrane. The membrane binding likely occurs through an interaction with lipid rafts, but is not required for F-actin assembly. Interesting the forced interaction of DAAM1 with plasma membrane through a rapamycin-dependent linkage, enhanced F-actin assembly at the cell membrane (compared to the cytoplasm) after the LatA washout. However, immediately after addition of both rapamycin and LatA, the cytoplasmic actin nodes formed transiently, before DAAM1 moved to the membrane. This was consistent with the idea that DAAM1 was initially anchored to cytoplasmic actin nodes. Further, photoactivatable tracking of DAAM1 showed DAAM1 was immobilized at these actin nodes. Thus, we suggest that DAAM1 organizes actin filaments into a nodal complex, and such nodal complexes seed actin network recovery after actin depolymerization. PMID:27760153

  15. Actin-binding proteins: the long road to understanding the dynamic landscape of cellular actin networks.

    PubMed

    Lappalainen, Pekka

    2016-08-15

    The actin cytoskeleton supports a vast number of cellular processes in nonmuscle cells. It is well established that the organization and dynamics of the actin cytoskeleton are controlled by a large array of actin-binding proteins. However, it was only 40 years ago that the first nonmuscle actin-binding protein, filamin, was identified and characterized. Filamin was shown to bind and cross-link actin filaments into higher-order structures and contribute to phagocytosis in macrophages. Subsequently many other nonmuscle actin-binding proteins were identified and characterized. These proteins regulate almost all steps of the actin filament assembly and disassembly cycles, as well as the arrangement of actin filaments into diverse three-dimensional structures. Although the individual biochemical activities of most actin-regulatory proteins are relatively well understood, knowledge of how these proteins function together in a common cytoplasm to control actin dynamics and architecture is only beginning to emerge. Furthermore, understanding how signaling pathways and mechanical cues control the activities of various actin-binding proteins in different cellular, developmental, and pathological processes will keep researchers busy for decades. PMID:27528696

  16. Integration of linear and dendritic actin nucleation in Nck-induced actin comets

    PubMed Central

    Borinskaya, Sofya; Velle, Katrina B.; Campellone, Kenneth G.; Talman, Arthur; Alvarez, Diego; Agaisse, Hervé; Wu, Yi I.; Loew, Leslie M.; Mayer, Bruce J.

    2016-01-01

    The Nck adaptor protein recruits cytosolic effectors such as N-WASP that induce localized actin polymerization. Experimental aggregation of Nck SH3 domains at the membrane induces actin comet tails—dynamic, elongated filamentous actin structures similar to those that drive the movement of microbial pathogens such as vaccinia virus. Here we show that experimental manipulation of the balance between unbranched/branched nucleation altered the morphology and dynamics of Nck-induced actin comets. Inhibition of linear, formin-based nucleation with the small-molecule inhibitor SMIFH2 or overexpression of the formin FH1 domain resulted in formation of predominantly circular-shaped actin structures with low mobility (actin blobs). These results indicate that formin-based linear actin polymerization is critical for the formation and maintenance of Nck-dependent actin comet tails. Consistent with this, aggregation of an exclusively branched nucleation-promoting factor (the VCA domain of N-WASP), with density and turnover similar to those of N-WASP in Nck comets, did not reconstitute dynamic, elongated actin comets. Furthermore, enhancement of branched Arp2/3-mediated nucleation by N-WASP overexpression caused loss of the typical actin comet tail shape induced by Nck aggregation. Thus the ratio of linear to dendritic nucleation activity may serve to distinguish the properties of actin structures induced by various viral and bacterial pathogens. PMID:26609071

  17. Integration of linear and dendritic actin nucleation in Nck-induced actin comets.

    PubMed

    Borinskaya, Sofya; Velle, Katrina B; Campellone, Kenneth G; Talman, Arthur; Alvarez, Diego; Agaisse, Hervé; Wu, Yi I; Loew, Leslie M; Mayer, Bruce J

    2016-01-15

    The Nck adaptor protein recruits cytosolic effectors such as N-WASP that induce localized actin polymerization. Experimental aggregation of Nck SH3 domains at the membrane induces actin comet tails--dynamic, elongated filamentous actin structures similar to those that drive the movement of microbial pathogens such as vaccinia virus. Here we show that experimental manipulation of the balance between unbranched/branched nucleation altered the morphology and dynamics of Nck-induced actin comets. Inhibition of linear, formin-based nucleation with the small-molecule inhibitor SMIFH2 or overexpression of the formin FH1 domain resulted in formation of predominantly circular-shaped actin structures with low mobility (actin blobs). These results indicate that formin-based linear actin polymerization is critical for the formation and maintenance of Nck-dependent actin comet tails. Consistent with this, aggregation of an exclusively branched nucleation-promoting factor (the VCA domain of N-WASP), with density and turnover similar to those of N-WASP in Nck comets, did not reconstitute dynamic, elongated actin comets. Furthermore, enhancement of branched Arp2/3-mediated nucleation by N-WASP overexpression caused loss of the typical actin comet tail shape induced by Nck aggregation. Thus the ratio of linear to dendritic nucleation activity may serve to distinguish the properties of actin structures induced by various viral and bacterial pathogens. PMID:26609071

  18. Xenopus egg cytoplasm with intact actin.

    PubMed

    Field, Christine M; Nguyen, Phuong A; Ishihara, Keisuke; Groen, Aaron C; Mitchison, Timothy J

    2014-01-01

    We report optimized methods for preparing Xenopus egg extracts without cytochalasin D, that we term "actin-intact egg extract." These are undiluted egg cytoplasm that contains abundant organelles, and glycogen which supplies energy, and represents the least perturbed cell-free cytoplasm preparation we know of. We used this system to probe cell cycle regulation of actin and myosin-II dynamics (Field et al., 2011), and to reconstitute the large, interphase asters that organize early Xenopus embryos (Mitchison et al., 2012; Wühr, Tan, Parker, Detrich, & Mitchison, 2010). Actin-intact Xenopus egg extracts are useful for analysis of actin dynamics, and interaction of actin with other cytoplasmic systems, in a cell-free system that closely mimics egg physiology, and more generally for probing the biochemistry and biophysics of the egg, zygote, and early embryo. Detailed protocols are provided along with assays used to check cell cycle state and tips for handling and storing undiluted egg extracts.

  19. Crystal structure of an archaeal actin homolog.

    PubMed

    Roeben, Annette; Kofler, Christine; Nagy, István; Nickell, Stephan; Hartl, F Ulrich; Bracher, Andreas

    2006-04-21

    Prokaryotic homologs of the eukaryotic structural protein actin, such as MreB and ParM, have been implicated in determination of bacterial cell shape, and in the segregation of genomic and plasmid DNA. In contrast to these bacterial actin homologs, little is known about the archaeal counterparts. As a first step, we expressed a predicted actin homolog of the thermophilic archaeon Thermoplasma acidophilum, Ta0583, and determined its crystal structure at 2.1A resolution. Ta0583 is expressed as a soluble protein in T.acidophilum and is an active ATPase at physiological temperature. In vitro, Ta0583 forms sheets with spacings resembling the crystal lattice, indicating an inherent propensity to form filamentous structures. The fold of Ta0583 contains the core structure of actin and clearly belongs to the actin/Hsp70 superfamily of ATPases. Ta0583 is approximately equidistant from actin and MreB on the structural level, and combines features from both eubacterial actin homologs, MreB and ParM. The structure of Ta0583 co-crystallized with ADP indicates that the nucleotide binds at the interface between the subdomains of Ta0583 in a manner similar to that of actin. However, the conformation of the nucleotide observed in complex with Ta0583 clearly differs from that in complex with actin, but closely resembles the conformation of ParM-bound nucleotide. On the basis of sequence and structural homology, we suggest that Ta0583 derives from a ParM-like actin homolog that was once encoded by a plasmid and was transferred into a common ancestor of Thermoplasma and Ferroplasma. Intriguingly, both genera are characterized by the lack of a cell wall, and therefore Ta0583 could have a function in cellular organization.

  20. Probing actin incorporation into myofibrils using Asp11 and His73 actin mutants.

    PubMed

    Xia, D; Peng, B; Sesok, D A; Peng, I

    1993-01-01

    We used a cell free system Bouché et al.: J. Cell Biol. 107:587-596, 1988] to study the incorporation of actin into myofibrils. We used alpha-skeletal muscle actin and actins with substitutions of either His73 [Solomon and Rubenstein: J. Biol.Chem. 262:11382, 1987], or Asp11 [Solomon et al.: J. Biol. Chem. 263:19662, 1988]. Actins were translated in reticulocyte lysate and incubated with myofibrils. The incorporated wild type actin could be cross-linked into dimers using N,N'-1,4-phenylenebismaleimide (PBM), indicating that the incorporated actin is actually inserted into the thin filaments of the myofibril. The His73 mutants incorporated to the same extent as wild type actin and was also cross-linked with PBM. Although some of the Asp11 mutants co-assembled with carrier actin, only 1-3% of the Asp11 mutant actins incorporated after 2 min and did not increase after 2 hr. Roughly 17% of wild type actin incorporated after 2 min and 31% after 2 hr. ATP increased the release of wild type actin from myofibrils, but did not increase the release of Asp11 mutants. We suggest that (1) the incorporation of wild type and His73 mutant actins was due to a physiological process whereas association of Asp11 mutants with myofibrils was non-specific, (2) the incorporation of wild type actin involved a rapid initial phase, followed by a slower phase, and (3) since some of the Asp11 mutants can co-assemble with wild type actin, the ability to self-assemble was not sufficient for incorporation into myofibrils. Thus, incorporation probably includes interaction between actin and a thin filament associated protein. We also showed that incorporation occurred at actin concentrations which would cause disassembly of F-actin. Since the myofibrils did not show large scale disassembly but incorporated actin, filament stability and monomer incorporation are likely to be mediated by actin associated proteins of the myofibril. PMID:8287497

  1. Dynamics of an actin spring

    NASA Astrophysics Data System (ADS)

    Riera, Christophe; Mahadevan, L.; Shin, Jennifer; Matsudaira, Paul

    2003-03-01

    The acrosome of the sperm of the horseshoe crab (Limulus Polyphemus) is an unusual actin based system that shows a spectacular dynamical transition in the presence of Ca++ that is present in abundance in the neighborhood of the egg. During this process, the bundle, which is initially bent and twisted uncoils and becomes straight in a matter of a few seconds. Based on microstructural data, we propose a model for the dynamics of uncoiling that is best represented by a triple-well potential corresponding to the different structural arrangements of the supertwisted filaments. Each of the false, true and coiled states corresponds to a local minimum of the energy, with the true state being the one with the lowest energy. Using an evolution equation derived by balancing torques, we investigate the nucleation and propagation of the phase transition and compare the results with those of experiments. Our model quantifies the hypothesis that the acrosomal bundle behaves like a mechano-chemical spring.

  2. Actin depolymerizing factor controls actin turnover and gliding motility in Toxoplasma gondii

    PubMed Central

    Mehta, Simren; Sibley, L. David

    2011-01-01

    Apicomplexan parasites rely on actin-based gliding motility to move across the substratum, cross biological barriers, and invade their host cells. Gliding motility depends on polymerization of parasite actin filaments, yet ∼98% of actin is nonfilamentous in resting parasites. Previous studies suggest that the lack of actin filaments in the parasite is due to inherent instability, leaving uncertain the role of actin-binding proteins in controlling dynamics. We have previously shown that the single allele of Toxoplasma gondii actin depolymerizing factor (TgADF) has strong actin monomer–sequestering and weak filament-severing activities in vitro. Here we used a conditional knockout strategy to investigate the role of TgADF in vivo. Suppression of TgADF led to accumulation of actin-rich filaments that were detected by immunofluorescence and electron microscopy. Parasites deficient in TgADF showed reduced speed of motility, increased aberrant patterns of motion, and inhibition of sustained helical gliding. Lack of TgADF also led to severe defects in entry and egress from host cells, thus blocking infection in vitro. These studies establish that the absence of stable actin structures in the parasite are not simply the result of intrinsic instability, but that TgADF is required for the rapid turnover of parasite actin filaments, gliding motility, and cell invasion. PMID:21346192

  3. Reversible stress softening of actin networks

    NASA Astrophysics Data System (ADS)

    Chaudhuri, Ovijit; Parekh, Sapun H.; Fletcher, Daniel A.

    2007-01-01

    The mechanical properties of cells play an essential role in numerous physiological processes. Organized networks of semiflexible actin filaments determine cell stiffness and transmit force during mechanotransduction, cytokinesis, cell motility and other cellular shape changes. Although numerous actin-binding proteins have been identified that organize networks, the mechanical properties of actin networks with physiological architectures and concentrations have been difficult to measure quantitatively. Studies of mechanical properties in vitro have found that crosslinked networks of actin filaments formed in solution exhibit stress stiffening arising from the entropic elasticity of individual filaments or crosslinkers resisting extension. Here we report reversible stress-softening behaviour in actin networks reconstituted in vitro that suggests a critical role for filaments resisting compression. Using a modified atomic force microscope to probe dendritic actin networks (like those formed in the lamellipodia of motile cells), we observe stress stiffening followed by a regime of reversible stress softening at higher loads. This softening behaviour can be explained by elastic buckling of individual filaments under compression that avoids catastrophic fracture of the network. The observation of both stress stiffening and softening suggests a complex interplay between entropic and enthalpic elasticity in determining the mechanical properties of actin networks.

  4. Actinic Granuloma with Focal Segmental Glomerulosclerosis

    PubMed Central

    Phasukthaworn, Ruedee; Chanprapaph, Kumutnart; Vachiramon, Vasanop

    2016-01-01

    Actinic granuloma is an uncommon granulomatous disease, characterized by annular erythematous plaque with central clearing predominately located on sun-damaged skin. The pathogenesis is not well understood, ultraviolet radiation is recognized as precipitating factor. We report a case of a 52-year-old woman who presented with asymptomatic annular erythematous plaques on the forehead and both cheeks persisting for 2 years. The clinical presentation and histopathologic findings support the diagnosis of actinic granuloma. During that period of time, she also developed focal segmental glomerulosclerosis. The association between actinic granuloma and focal segmental glomerulosclerosis needs to be clarified by further studies. PMID:27293392

  5. Binding of actin to lens alpha crystallins

    NASA Technical Reports Server (NTRS)

    Gopalakrishnan, S.; Takemoto, L.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    Actin has been coupled to a cyanogen bromide-activated Sepharose 4B column, then tested for binding to alpha, beta, and gamma crystallin preparations from the bovine lens. Alpha, but not beta or gamma, crystallins bound to the actin affinity column in a time dependent and saturable manner. Subfractionation of the alpha crystallin preparation into the alpha-A and alpha-B species, followed by incubation with the affinity column, demonstrated that both species bound approximately the same. Together, these studies demonstrate a specific and saturable binding of lens alpha-A and alpha-B with actin.

  6. Dynamic reorganization of the actin cytoskeleton

    PubMed Central

    Gressin, Laurène; Théry, Manuel; Blanchoin, Laurent

    2015-01-01

    Cellular processes, including morphogenesis, polarization, and motility, rely on a variety of actin-based structures. Although the biochemical composition and filament organization of these structures are different, they often emerge from a common origin. This is possible because the actin structures are highly dynamic. Indeed, they assemble, grow, and disassemble in a time scale of a second to a minute. Therefore, the reorganization of a given actin structure can promote the formation of another. Here, we discuss such transitions and illustrate them with computer simulations. PMID:26989473

  7. Computational model of polarized actin cables and cytokinetic actin ring formation in budding yeast

    PubMed Central

    Tang, Haosu; Bidone, Tamara C.

    2015-01-01

    The budding yeast actin cables and contractile ring are important for polarized growth and division, revealing basic aspects of cytoskeletal function. To study these formin-nucleated structures, we built a 3D computational model with actin filaments represented as beads connected by springs. Polymerization by formins at the bud tip and bud neck, crosslinking, severing, and myosin pulling, are included. Parameter values were estimated from prior experiments. The model generates actin cable structures and dynamics similar to those of wild type and formin deletion mutant cells. Simulations with increased polymerization rate result in long, wavy cables. Simulated pulling by type V myosin stretches actin cables. Increasing the affinity of actin filaments for the bud neck together with reduced myosin V pulling promotes the formation of a bundle of antiparallel filaments at the bud neck, which we suggest as a model for the assembly of actin filaments to the contractile ring. PMID:26538307

  8. Correlation between polymerizability and conformation in scallop beta-like actin and rabbit skeletal muscle alpha-actin.

    PubMed

    Khaitlina, S; Antropova, O; Kuznetsova, I; Turoverov, K; Collins, J H

    1999-08-01

    In order to investigate the structural basis for functional differences among actin isoforms, we have compared the polymerization properties and conformations of scallop adductor muscle beta-like actin and rabbit skeletal muscle alpha-actin. Polymerization of scallop Ca(2+)-actin was slower than that of skeletal muscle Ca(2+)-actin. Cleavage of the actin polypeptide chain between Gly-42 and Val-43 with Escherichia coli protease ECP 32 impaired the polymerization of scallop Mg(2+)-actin to a greater extent than skeletal muscle Mg(2+)-actin. When monomeric scallop and skeletal muscle Ca(2+)-actins were subjected to limited proteolysis with trypsin, subtilisin, or ECP 32, no differences in the conformation of actin subdomain 2 were detected. At the same time, local differences in the conformations of scallop and skeletal muscle actin subdomains 1 were revealed as intrinsic fluorescence differences. Replacement of tightly bound Ca(2+) with Mg(2+) resulted in more extensive proteolysis of segment 61-69 of scallop actin than in the case of skeletal muscle actin. Furthermore, segment 61-69 was more accessible to proteolysis with subtilisin in polymerized scallop Ca(2+)-actin than in polymerized skeletal muscle Ca(2+)-actin, indicating that, in the polymeric form, the nucleotide-containing cleft is in a more open conformation in beta-like scallop actin than in skeletal muscle alpha-actin. We suggest that this difference between scallop and skeletal muscle actins is due to a less efficient shift of scallop actin subdomain 2 to the position it has in the polymer. The possible consequences of amino acid substitutions in actin subdomain 1 in the allosteric regulation of the actin cleft, and hence in the different stabilities of polymers formed by different actins, are discussed. PMID:10415117

  9. Structural Differences Explain Diverse Functions of Plasmodium Actins

    PubMed Central

    Vahokoski, Juha; Martinez, Silvia Muñico; Ignatev, Alexander; Lepper, Simone; Frischknecht, Friedrich; Sidén-Kiamos, Inga; Sachse, Carsten; Kursula, Inari

    2014-01-01

    Actins are highly conserved proteins and key players in central processes in all eukaryotic cells. The two actins of the malaria parasite are among the most divergent eukaryotic actins and also differ from each other more than isoforms in any other species. Microfilaments have not been directly observed in Plasmodium and are presumed to be short and highly dynamic. We show that actin I cannot complement actin II in male gametogenesis, suggesting critical structural differences. Cryo-EM reveals that Plasmodium actin I has a unique filament structure, whereas actin II filaments resemble canonical F-actin. Both Plasmodium actins hydrolyze ATP more efficiently than α-actin, and unlike any other actin, both parasite actins rapidly form short oligomers induced by ADP. Crystal structures of both isoforms pinpoint several structural changes in the monomers causing the unique polymerization properties. Inserting the canonical D-loop to Plasmodium actin I leads to the formation of long filaments in vitro. In vivo, this chimera restores gametogenesis in parasites lacking actin II, suggesting that stable filaments are required for exflagellation. Together, these data underline the divergence of eukaryotic actins and demonstrate how structural differences in the monomers translate into filaments with different properties, implying that even eukaryotic actins have faced different evolutionary pressures and followed different paths for developing their polymerization properties. PMID:24743229

  10. Actin filament organization of foot processes in vertebrate glomerular podocytes.

    PubMed

    Ichimura, Koichiro; Kurihara, Hidetake; Sakai, Tatsuo

    2007-09-01

    We investigated the actin filament organization and immunolocalization of actin-binding proteins (alpha-actinin and cortactin) in the podocyte foot processes of eight vertebrate species (lamprey, carp, newt, frog, gecko, turtle, quail, and rat). Three types of actin cytoskeleton were found in these foot processes. (1) A cortical actin network with cortactin filling the space between the plasma membrane and the other actin cytoskeletons described below was found in all of the species examined here. The data indicated that the cortical actin network was the minimal essential actin cytoskeleton for the formation and maintenance of the foot processes in vertebrate podocytes. (2) An actin bundle with alpha-actinin existing along the longitudinal axis of foot process above the level of slit diaphragms was only observed in quail and rat. (3) An actin fascicle consisting of much fewer numbers of actin filaments than that of the actin bundle was observed in the species other than quail and rat, but at various frequencies. These findings suggest that the actin bundle is an additional actin cytoskeleton reflecting a functional state peculiar to quail and rat glomeruli. Considering the higher intraglomerular pressure and the extremely thin filtration barrier in birds and mammals, the foot processes probably mainly protect the thinner filtration barrier from the higher internal pressure occurring in quail and rat glomeruli. Therefore, we consider that the actin bundle plays a crucial role in the mechanical protection of the filtration barrier. Moreover, the actin fascicle may be a potential precursor of the actin bundle.

  11. Genetics Home Reference: actin-accumulation myopathy

    MedlinePlus

    ... 7(3):160-8. Citation on PubMed Laing NG, Dye DE, Wallgren-Pettersson C, Richard G, Monnier ... Vigneron J, Wallgren-Pettersson C, Beggs AH, Laing NG. Mutations in the skeletal muscle alpha-actin gene ...

  12. [Actin in the wound healing process].

    PubMed

    Nowak, Dorota; Popow-Woźniak, Agnieszka; Raźnikiewicz, Linda; Malicka-Błaszkiewicz, Maria

    2009-01-01

    Wound healing is an important biological process of crucial value for organisms survival and retention of its proper functions. The recognition of molecular mechanisms of these phenomenon is still under investigation. The transition of mesenchymal fibroblasts to myofibroblasts is a key point in wound healing. The contraction ability of myofibroblast enables the shrinkage of a wound and closes its edges. Alpha smooth muscle actin (alpha-SMA), one of six actin isoforms, is a marker of compeletely differentiated myofibroblast. The regulation of differentiation process depends on many growth factors (especially TGF beta 1), the level of active thymosin beta 4, extracellular matrix proteins--including fibronectin, and also on specificity of microenvironment. Thymosin beta 4 is responsible for maintenance of pool of monomeric actin and actin filaments depolymerization. It can also act as a transcription factor, migration stimulator and immunomodulator, so this protein deserves for more attention in wound healing research field. PMID:19824469

  13. Structural Dynamics of an Actin Spring

    PubMed Central

    Mahadevan, L.; Riera, C.S.; Shin, Jennifer H.

    2011-01-01

    Actin-based motility in cells is usually associated with either polymerization/depolymerization in the presence of cross-linkers or contractility in the presence of myosin motors. Here, we focus on a third distinct mechanism involving actin in motility, seen in the dynamics of an active actin spring that powers the acrosomal reaction of the horseshoe crab (Limulus polyphemus) sperm. During this process, a 60-μm bent and twisted bundle of cross-linked actin uncoils and becomes straight in a few seconds in the presence of Ca2+. This straightening, which occurs at a constant velocity, allows the acrosome to forcefully penetrate the egg. Synthesizing ultrastructural information with the kinetics, energetics, and imaging of calcium binding allows us to construct a dynamical theory for this mechanochemical engine consistent with our experimental observations. It also illuminates the general mechanism by which energy may be stored in conformational changes and released cooperatively in ordered macromolecular assemblies. PMID:21320427

  14. Mechanics model for actin-based motility.

    PubMed

    Lin, Yuan

    2009-02-01

    We present here a mechanics model for the force generation by actin polymerization. The possible adhesions between the actin filaments and the load surface, as well as the nucleation and capping of filament tips, are included in this model on top of the well-known elastic Brownian ratchet formulation. A closed form solution is provided from which the force-velocity relationship, summarizing the mechanics of polymerization, can be drawn. Model predictions on the velocity of moving beads driven by actin polymerization are consistent with experiment observations. This model also seems capable of explaining the enhanced actin-based motility of Listeria monocytogenes and beads by the presence of Vasodilator-stimulated phosphoprotein, as observed in recent experiments.

  15. Mechanics model for actin-based motility

    NASA Astrophysics Data System (ADS)

    Lin, Yuan

    2009-02-01

    We present here a mechanics model for the force generation by actin polymerization. The possible adhesions between the actin filaments and the load surface, as well as the nucleation and capping of filament tips, are included in this model on top of the well-known elastic Brownian ratchet formulation. A closed form solution is provided from which the force-velocity relationship, summarizing the mechanics of polymerization, can be drawn. Model predictions on the velocity of moving beads driven by actin polymerization are consistent with experiment observations. This model also seems capable of explaining the enhanced actin-based motility of Listeria monocytogenes and beads by the presence of Vasodilator-stimulated phosphoprotein, as observed in recent experiments.

  16. Nematic textures in F-actin

    NASA Astrophysics Data System (ADS)

    Das, P.; Roy, J.; Chakrabarti, N.; Basu, S.; Das, U.

    2002-05-01

    Actin filaments, which are protein polymers occurring abundantly and ubiquitously in muscle and nonmuscle cells, are known to align in a shear flow, and with an external magnetic field. They form a nematic liquid crystal of the athermal type at a low concentration. Typical defects and textures of the nematic actin liquid crystal are described in this work. The generation of well-aligned nematic single crystals has been reported, in the vicinity of an air-water interface, with the actin filaments spontaneously aligning normal to the interface. Away from the air-water interface nematic single crystal domains are due to the alignment of the actin filaments parallel to the glass surface. The twist-bend nature of the disclination line of integral strength (m=1) has been attributed to the relative magnitudes of the anisotropic curvature elastic constants, which reflect the filaments' semirigidity.

  17. Actinic review of EUV masks

    NASA Astrophysics Data System (ADS)

    Feldmann, Heiko; Ruoff, Johannes; Harnisch, Wolfgang; Kaiser, Winfried

    2010-04-01

    Management of mask defects is a major challenge for the introduction of EUV for HVM production. Once a defect has been detected, its printing impact needs to be predicted. Potentially the defect requires some repair, the success of which needs to be proven. This defect review has to be done with an actinic inspection system that matches the imaging conditions of an EUV scanner. During recent years, several concepts for such an aerial image metrology system (AIMS™) have been proposed. However, until now no commercial solution exists for EUV. Today, advances in EUV optics technology allow envisioning a solution that has been discarded before as unrealistic. We present this concept and its technical cornerstones.While the power requirement for the EUV source is less demanding than for HVM lithography tools, radiance, floor space, and stability are the main criteria for source selection. The requirement to emulate several generations of EUV scanners demands a large flexibility for the ilumination and imaging systems. New critical specifications to the EUV mirrors in the projection microscope can be satisfied using our expertise from lithographic mirrors. In summary, an EUV AIMS™ meeting production requirements seems to be feasible.

  18. Elasticity of F-actin networks

    NASA Astrophysics Data System (ADS)

    Gardel, Margaret Lise

    This thesis presents a study of the elasticity and microstructure of three filamentous actin (F-actin) based materials. Using bulk rheology, microrheology, multiple particle tracking and imaging techniques, we study the microscopic origins of the mechanical properties of F-actin networks. We briefly introduce aspects of F-actin and rheology essential to provide a background for and motivate this thesis in Chapter 1. In Chapter 2, we describe the materials and methods used. An introduction to microrheology is given in Chapter 3. In Chapter 4, we study solutions of entangled F-actin. We elucidate the microscopic origins of bulk elasticity using microrheology techniques. We also show that multiple particle tracking can also probe the dynamics of the F-actin solution microstructure. We explore the effect of rigid, incompliant chemical cross-links between actin filaments in Chapter 5. We explore changes in the network microstructure as the concentration of cross-links is varied. We find that the elastic stiffness of these networks is extremely sensitive to small changes in cross-link density. Despite this large variation, the linear viscoelasticity of all networks can be scaled onto a universal master curve; this scaling reveals that the mechanical dissipation of the networks is due to thermal fluctuations of F-actin. At large stresses, the mechanical stiffness of these networks diverges. The form of this stress stiffening response is consistent with the non-linear force extension of a single semi-flexible polymer. Thus, over a large range of conditions, the linear and nonlinear mechanical response of rigidly cross-linked networks is entropic in origin. Finally, at very low cross-link and filament densities, we observe a transition to a qualitatively different type of elasticity; this is consistent with a transition to an enthalpic network elasticity dominated by bending of F-actin. In Chapter 6, we study the elastic properties of F-actin networks assembled with a

  19. Mechanism of Actin Filament Bundling by Fascin

    SciTech Connect

    Jansen, Silvia; Collins, Agnieszka; Yang, Changsong; Rebowski, Grzegorz; Svitkina, Tatyana; Dominguez, Roberto

    2013-03-07

    Fascin is the main actin filament bundling protein in filopodia. Because of the important role filopodia play in cell migration, fascin is emerging as a major target for cancer drug discovery. However, an understanding of the mechanism of bundle formation by fascin is critically lacking. Fascin consists of four {beta}-trefoil domains. Here, we show that fascin contains two major actin-binding sites, coinciding with regions of high sequence conservation in {beta}-trefoil domains 1 and 3. The site in {beta}-trefoil-1 is located near the binding site of the fascin inhibitor macroketone and comprises residue Ser-39, whose phosphorylation by protein kinase C down-regulates actin bundling and formation of filopodia. The site in {beta}-trefoil-3 is related by pseudo-2-fold symmetry to that in {beta}-trefoil-1. The two sites are {approx}5 nm apart, resulting in a distance between actin filaments in the bundle of {approx}8.1 nm. Residue mutations in both sites disrupt bundle formation in vitro as assessed by co-sedimentation with actin and electron microscopy and severely impair formation of filopodia in cells as determined by rescue experiments in fascin-depleted cells. Mutations of other areas of the fascin surface also affect actin bundling and formation of filopodia albeit to a lesser extent, suggesting that, in addition to the two major actin-binding sites, fascin makes secondary contacts with other filaments in the bundle. In a high resolution crystal structure of fascin, molecules of glycerol and polyethylene glycol are bound in pockets located within the two major actin-binding sites. These molecules could guide the rational design of new anticancer fascin inhibitors.

  20. Actin: its cumbersome pilgrimage through cellular compartments.

    PubMed

    Schleicher, Michael; Jockusch, Brigitte M

    2008-06-01

    In this article, we follow the history of one of the most abundant, most intensely studied proteins of the eukaryotic cells: actin. We report on hallmarks of its discovery, its structural and functional characterization and localization over time, and point to present days' knowledge on its position as a member of a large family. We focus on the rather puzzling number of diverse functions as proposed for actin as a dual compartment protein. Finally, we venture on some speculations as to its origin.

  1. Sarcomeric Pattern Formation by Actin Cluster Coalescence

    PubMed Central

    Friedrich, Benjamin M.; Fischer-Friedrich, Elisabeth; Gov, Nir S.; Safran, Samuel A.

    2012-01-01

    Contractile function of striated muscle cells depends crucially on the almost crystalline order of actin and myosin filaments in myofibrils, but the physical mechanisms that lead to myofibril assembly remains ill-defined. Passive diffusive sorting of actin filaments into sarcomeric order is kinetically impossible, suggesting a pivotal role of active processes in sarcomeric pattern formation. Using a one-dimensional computational model of an initially unstriated actin bundle, we show that actin filament treadmilling in the presence of processive plus-end crosslinking provides a simple and robust mechanism for the polarity sorting of actin filaments as well as for the correct localization of myosin filaments. We propose that the coalescence of crosslinked actin clusters could be key for sarcomeric pattern formation. In our simulations, sarcomere spacing is set by filament length prompting tight length control already at early stages of pattern formation. The proposed mechanism could be generic and apply both to premyofibrils and nascent myofibrils in developing muscle cells as well as possibly to striated stress-fibers in non-muscle cells. PMID:22685394

  2. Resemblance of actin-binding protein/actin gels to covalently crosslinked networks

    NASA Astrophysics Data System (ADS)

    Janmey, Paul A.; Hvidt, Søren; Lamb, Jennifer; Stossel, Thomas P.

    1990-05-01

    THE maintainance of the shape of cells is often due to their surface elasticity, which arises mainly from an actin-rich cytoplasmic cortex1,2. On locomotion, phagocytosis or fission, however, these cells become partially fluid-like. The finding of proteins that can bind to actin and control the assembly of, or crosslink, actin filaments, and of intracellular messages that regulate the activities of some of these actin-binding proteins, indicates that such 'gel sol' transformations result from the rearrangement of cortical actin-rich networks3. Alternatively, on the basis of a study of the mechanical properties of mixtures of actin filaments and an Acanthamoeba actin-binding protein, α-actinin, it has been proposed that these transformations can be accounted for by rapid exchange of crosslinks between actin filaments4: the cortical network would be solid when the deformation rate is greater than the rate of crosslink exchange, but would deform or 'creep' when deformation is slow enough to permit crosslinker molecules to rearrange. Here we report, however, that mixtures of actin filaments and actin-binding protein (ABP), an actin crosslinking protein of many higher eukaryotes, form gels Theologically equivalent to covalently crosslinked networks. These gels do not creep in response to applied stress on a time scale compatible with most cell-surface movements. These findings support a more complex and controlled mechanism underlying the dynamic mechanical properties of cortical cytoplasm, and can explain why cells do not collapse under the constant shear forces that often exist in tissues.

  3. Nuclear and cytoplasmic actin in dinoflagellates.

    PubMed

    Soyer-Gobillard, M O; Ausseil, J; Géraud, M L

    1996-01-01

    Experiments using monoclonal and polyclonal anti-actin antibodies allowed us to demonstrate the presence of F- or G-actin in original protists, dinoflagellates, either by biochemistry, immunofluorescence and in TEM. SDS-PAGE electrophoresis and immunoblottings made either from total or nuclear protein extracts revealed the presence of a 44-kDa band reacting with monoclonal anti-actin antibody in two species, Prorocentrum micans and Crypthecodinium cohnii, and thus demonstrated the presence of actin in nuclear and cytoplasmic fractions. After squash preparation of P micans cells, actin was identified within the nucleus and in some regions of the cytoplasm by immunofluorescence microscopy. Labelling of both the nucleolus and the centrosome region was evident together with amorphous nucleoplasmic material surrounding the chromosomes. The use of cryosections of intact P micans and C cohnii cells for immunofluorescence along with staining with DAPI to delineate the chromosomes themselves, yielded finer resolution of the intranuclear network labelling pattern and allowed us to complete our observations, in particular on the cytoplasmic labelling. In P micans, in addition to the centrosome region, the cytoplasmic channels passing through the nucleus in dividing cells are labelled. In C cohnii, the cortex, the centrosome region, the cytoplasmic channels, the region surrounding the nucleus, the filaments linking it to the cortex and the cleavage furrow are also labelled. In the nucleus of the two species, there is a prominent "weft' of fine actin filaments in the nucleoplasm forming a matrix of varying density around the persistent chromosomes. This actin matrix, of unknown function, is most conspicuous at the end of the S-phase of the cell cycle. Fluorescent derivatives of phalloidin, used as diagnostic cytochemical probes for polymeric actin (F-actin), gave similar results. Positive TEM immunolabelling of intranuclear actin confirms its presence in the nucleoplasm, in the

  4. Interactions of actin, myosin, and an actin-binding protein of chronic myelogenous leukemia leukocytes.

    PubMed Central

    Boxer, L A; Stossel, T P

    1976-01-01

    Actin, myosin, and a high molecular weight actin-binding protein were purified from chronic myelogenous leukemia (CML) leukocytes. CML leukocyte actin resembled skeletal muscle and other cytoplasmic actins by its subunit molecular weight, by its ability to polymerize in the presence of salts, and to activate the Mg2+-ATPase activity of rabbit skeletal muscle myosin. CML leukocyte myosin was similar to other vertebrate cytoplasmic myosins in having heavy chains and two light subunits. However, its apparent heavy-chain molecular weight and Stokes radius suggested that it was variably degraded during purification. Purified CML leukocyte myosin had average specific EDTA- AND Ca2+-activated ATPase activities of 125 and 151 nmol Pi released/mg protein per min, respectively and low specific Mg2+-ATPase activity. The Mg2+-ATPase activity of CML myosin was increased 200-fold by rabbit skeletal muscle F-actin, but the specific activity relative to that of actin-activated rabbit skeletal muscle myosin was low. CML leukocyte myosin, like other vertebrate cytoplasmic myosins, formed filaments in 0.1 M KCl solutions. Reduced and denatured CML leukocyte-actin-binding protein had a single high molecular weight subunit like a recently described actin-binding protein of rabbit pulmonary macrophages which promotes the polymerization and gelation of actin. Cytoplasmic extracts of CML leukocytes prepared with ice-cold 0.34-M sucrose solutions containing Mg2+-ATP, dithiothreitol, and EDTA at pH 7.0 underwent rapid gelation when warmed to 25 degrees C. Initially, the gel could be liquified by cooling to ice-bath temperature. With time, warmed cytoplasmic extract gels shrunk ("contracted") into aggregates. The following findings indicated that CML leukocyte actin-binding protein promoted the temperature-dependent gelation of actin in the cytoplasmic extracts and that CML leukocyte myosin was involved in the contraction of the actin gels: (a) Cytoplasmic extract gels initially contained

  5. Defining a core set of actin cytoskeletal proteins critical for actin-based motility of Rickettsia.

    PubMed

    Serio, Alisa W; Jeng, Robert L; Haglund, Cat M; Reed, Shawna C; Welch, Matthew D

    2010-05-20

    Many Rickettsia species are intracellular bacterial pathogens that use actin-based motility for spread during infection. However, while other bacteria assemble actin tails consisting of branched networks, Rickettsia assemble long parallel actin bundles, suggesting the use of a distinct mechanism for exploiting actin. To identify the underlying mechanisms and host factors involved in Rickettsia parkeri actin-based motility, we performed an RNAi screen targeting 115 actin cytoskeletal genes in Drosophila cells. The screen delineated a set of four core proteins-profilin, fimbrin/T-plastin, capping protein, and cofilin--as crucial for determining actin tail length, organizing filament architecture, and enabling motility. In mammalian cells, these proteins were localized throughout R. parkeri tails, consistent with a role in motility. Profilin and fimbrin/T-plastin were critical for the motility of R. parkeri but not Listeria monocytogenes. Our results highlight key distinctions between the evolutionary strategies and molecular mechanisms employed by bacterial pathogens to assemble and organize actin. PMID:20478540

  6. SelR reverses Mical-mediated oxidation of actin to regulate F-actin dynamics.

    PubMed

    Hung, Ruei-Jiun; Spaeth, Christopher S; Yesilyurt, Hunkar Gizem; Terman, Jonathan R

    2013-12-01

    Actin's polymerization properties are markedly altered by oxidation of its conserved Met 44 residue. Mediating this effect is a specific oxidation-reduction (redox) enzyme, Mical, that works with Semaphorin repulsive guidance cues and selectively oxidizes Met 44. We now find that this actin-regulatory process is reversible. Employing a genetic approach, we identified a specific methionine sulfoxide reductase (MsrB) enzyme SelR that opposes Mical redox activity and Semaphorin-Plexin repulsion to direct multiple actin-dependent cellular behaviours in vivo. SelR specifically catalyses the reduction of the R isomer of methionine sulfoxide (methionine-R-sulfoxide) to methionine, and we found that SelR directly reduced Mical-oxidized actin, restoring its normal polymerization properties. These results indicate that Mical oxidizes actin stereospecifically to generate actin Met-44-R-sulfoxide (actin(Met(R)O-44)), and also implicate the interconversion of specific Met/Met(R)O residues as a precise means to modulate protein function. Our results therefore uncover a specific reversible redox actin regulatory system that controls cell and developmental biology.

  7. Lamellipodin promotes actin assembly by clustering Ena/VASP proteins and tethering them to actin filaments.

    PubMed

    Hansen, Scott D; Mullins, R Dyche

    2015-01-01

    Enabled/Vasodilator (Ena/VASP) proteins promote actin filament assembly at multiple locations, including: leading edge membranes, focal adhesions, and the surface of intracellular pathogens. One important Ena/VASP regulator is the mig-10/Lamellipodin/RIAM family of adaptors that promote lamellipod formation in fibroblasts and drive neurite outgrowth and axon guidance in neurons. To better understand how MRL proteins promote actin network formation we studied the interactions between Lamellipodin (Lpd), actin, and VASP, both in vivo and in vitro. We find that Lpd binds directly to actin filaments and that this interaction regulates its subcellular localization and enhances its effect on VASP polymerase activity. We propose that Lpd delivers Ena/VASP proteins to growing barbed ends and increases their polymerase activity by tethering them to filaments. This interaction represents one more pathway by which growing actin filaments produce positive feedback to control localization and activity of proteins that regulate their assembly.

  8. Septins promote F-actin ring formation by crosslinking actin filaments into curved bundles.

    PubMed

    Mavrakis, Manos; Azou-Gros, Yannick; Tsai, Feng-Ching; Alvarado, José; Bertin, Aurélie; Iv, Francois; Kress, Alla; Brasselet, Sophie; Koenderink, Gijsje H; Lecuit, Thomas

    2014-04-01

    Animal cell cytokinesis requires a contractile ring of crosslinked actin filaments and myosin motors. How contractile rings form and are stabilized in dividing cells remains unclear. We address this problem by focusing on septins, highly conserved proteins in eukaryotes whose precise contribution to cytokinesis remains elusive. We use the cleavage of the Drosophila melanogaster embryo as a model system, where contractile actin rings drive constriction of invaginating membranes to produce an epithelium in a manner akin to cell division. In vivo functional studies show that septins are required for generating curved and tightly packed actin filament networks. In vitro reconstitution assays show that septins alone bundle actin filaments into rings, accounting for the defects in actin ring formation in septin mutants. The bundling and bending activities are conserved for human septins, and highlight unique functions of septins in the organization of contractile actomyosin rings.

  9. Arabidopsis ACTIN-DEPOLYMERIZING FACTOR7 Severs Actin Filaments and Regulates Actin Cable Turnover to Promote Normal Pollen Tube Growth[W

    PubMed Central

    Zheng, Yiyan; Xie, Yurong; Jiang, Yuxiang; Qu, Xiaolu; Huang, Shanjin

    2013-01-01

    Actin filaments are often arranged into higher-order structures, such as the longitudinal actin cables that generate the reverse fountain cytoplasmic streaming pattern present in pollen tubes. While several actin binding proteins have been implicated in the generation of these cables, the mechanisms that regulate their dynamic turnover remain largely unknown. Here, we show that Arabidopsis thaliana ACTIN-DEPOLYMERIZING FACTOR7 (ADF7) is required for turnover of longitudinal actin cables. In vitro biochemical analyses revealed that ADF7 is a typical ADF that prefers ADP-G-actin over ATP-G-actin. ADF7 inhibits nucleotide exchange on actin and severs filaments, but its filament severing and depolymerizing activities are less potent than those of the vegetative ADF1. ADF7 primarily decorates longitudinal actin cables in the shanks of pollen tubes. Consistent with this localization pattern, the severing frequency and depolymerization rate of filaments significantly decreased, while their maximum lifetime significantly increased, in adf7 pollen tube shanks. Furthermore, an ADF7–enhanced green fluorescent protein fusion with defective severing activity but normal G-actin binding activity could not complement adf7, providing compelling evidence that the severing activity of ADF7 is vital for its in vivo functions. These observations suggest that ADF7 evolved to promote turnover of longitudinal actin cables by severing actin filaments in pollen tubes. PMID:24058157

  10. Purification and Characterization of Actin from Maize Pollen 1

    PubMed Central

    Liu, Xiong; Yen, Lung-Fei

    1992-01-01

    Pollen is an excellent source of actin for biochemical and physiological studies of the actomyosin system in higher plants. We have developed an efficient method to prepare relatively high levels of actin from the pollen of maize (Zea mays L.). The procedures of purification include acetone powder preparation, saturated ammonium sulfate fractionation, diethylaminoethyl-cellulose chromatography, a cycle of polymerization-depolymerization, and Sephacryl S-200 gel filtration. The average yield of actin is 19 milligrams per 100 grams of pollen grains extracted. This is comparable with those of Acanthamoeba castellanii and human platelets. The purified pollen actin is electrophoretically homogeneous and its molecular mass is 42 kilodaltons. The amino acid composition and circular dichroism spectrum of pollen actin are identical to those of muscle actin. The actin purified from pollen is able to polymerize to F-actin. The pollen F-actin activated the activity of the muscle myosin ATPase sevenfold. ImagesFigure 1Figure 2 PMID:16668982

  11. Tau co-organizes dynamic microtubule and actin networks

    PubMed Central

    Elie, Auréliane; Prezel, Elea; Guérin, Christophe; Denarier, Eric; Ramirez-Rios, Sacnicte; Serre, Laurence; Andrieux, Annie; Fourest-Lieuvin, Anne; Blanchoin, Laurent; Arnal, Isabelle

    2015-01-01

    The crosstalk between microtubules and actin is essential for cellular functions. However, mechanisms underlying the microtubule-actin organization by cross-linkers remain largely unexplored. Here, we report that tau, a neuronal microtubule-associated protein, binds to microtubules and actin simultaneously, promoting in vitro co-organization and coupled growth of both networks. By developing an original assay to visualize concomitant microtubule and actin assembly, we show that tau can induce guided polymerization of actin filaments along microtubule tracks and growth of single microtubules along actin filament bundles. Importantly, tau mediates microtubule-actin co-alignment without changing polymer growth properties. Mutagenesis studies further reveal that at least two of the four tau repeated motifs, primarily identified as tubulin-binding sites, are required to connect microtubules and actin. Tau thus represents a molecular linker between microtubule and actin networks, enabling a coordination of the two cytoskeletons that might be essential in various neuronal contexts. PMID:25944224

  12. Sensing actin dynamics: Structural basis for G-actin-sensitive nuclear import of MAL

    SciTech Connect

    Hirano, Hidemi; Matsuura, Yoshiyuki

    2011-10-22

    Highlights: {yields} MAL has a bipartite NLS that binds to Imp{alpha} in an extended conformation. {yields} Mutational analyses verified the functional significance of MAL-Imp{alpha} interactions. {yields} Induced folding and NLS-masking by G-actins inhibit nuclear import of MAL. -- Abstract: The coordination of cytoskeletal actin dynamics with gene expression reprogramming is emerging as a crucial mechanism to control diverse cellular processes, including cell migration, differentiation and neuronal circuit assembly. The actin-binding transcriptional coactivator MAL (also known as MRTF-A/MKL1/BSAC) senses G-actin concentration and transduces Rho GTPase signals to serum response factor (SRF). MAL rapidly shuttles between the cytoplasm and the nucleus in unstimulated cells but Rho-induced depletion of G-actin leads to MAL nuclear accumulation and activation of transcription of SRF:MAL-target genes. Although the molecular and structural basis of actin-regulated nucleocytoplasmic shuttling of MAL is not understood fully, it is proposed that nuclear import of MAL is mediated by importin {alpha}/{beta} heterodimer, and that G-actin competes with importin {alpha}/{beta} for the binding to MAL. Here we present structural, biochemical and cell biological evidence that MAL has a classical bipartite nuclear localization signal (NLS) in the N-terminal 'RPEL' domain containing Arg-Pro-X-X-X-Glu-Leu (RPEL) motifs. The NLS residues of MAL adopt an extended conformation and bind along the surface groove of importin-{alpha}, interacting with the major- and minor-NLS binding sites. We also present a crystal structure of wild-type MAL RPEL domain in complex with five G-actins. Comparison of the importin-{alpha}- and actin-complexes revealed that the binding of G-actins to MAL is associated with folding of NLS residues into a helical conformation that is inappropriate for importin-{alpha} recognition.

  13. Incorporation of mammalian actin into microfilaments in plant cell nucleus

    PubMed Central

    Paves, Heiti; Truve, Erkki

    2004-01-01

    Background Actin is an ancient molecule that shows more than 90% amino acid homology between mammalian and plant actins. The regions of the actin molecule that are involved in F-actin assembly are largely conserved, and it is likely that mammalian actin is able to incorporate into microfilaments in plant cells but there is no experimental evidence until now. Results Visualization of microfilaments in onion bulb scale epidermis cells by different techniques revealed that rhodamine-phalloidin stained F-actin besides cytoplasm also in the nuclei whereas GFP-mouse talin hybrid protein did not enter the nuclei. Microinjection of fluorescently labeled actin was applied to study the presence of nuclear microfilaments in plant cells. Ratio imaging of injected fluorescent rabbit skeletal muscle actin and phalloidin staining of the microinjected cells showed that mammalian actin was able to incorporate into plant F-actin. The incorporation occurred preferentially in the nucleus and in the perinuclear region of plant cells whereas part of plant microfilaments, mostly in the periphery of cytoplasm, did not incorporate mammalian actin. Conclusions Microinjected mammalian actin is able to enter plant cell's nucleus, whereas incorporation of mammalian actin into plant F-actin occurs preferentially in the nucleus and perinuclear area. PMID:15102327

  14. The EGF receptor is an actin-binding protein

    PubMed Central

    1992-01-01

    In a number of recent studies it has been shown that in vivo part of the EGF receptor (EGFR) population is associated to the actin filament system. In this paper we demonstrate that the purified EGFR can be cosedimented with purified filamentous actin (F-actin) indicating a direct association between EGFR and actin. A truncated EGFR, previously shown not to be associated to the cytoskeleton, was used as a control and this receptor did not cosediment with actin filaments. Determination of the actin-binding domain of the EGFR was done by measuring competition of either a polyclonal antibody or synthetic peptides on EGFR cosedimentation with F-actin. A synthetic peptide was made homologous to amino acid residues 984-996 (HL-33) of the EGFR which shows high homology with the actin-binding domain of Acanthamoeba profilin. A polyclonal antibody raised against HL-33 was found to prevent cosedimentation of EGFR with F-actin. This peptide HL-33 was shown to bind directly to actin in contrast with a synthetic peptide homologous to residues 1001-1013 (HL-34). During cosedimentation, HL-33 competed for actin binding of the EGFR and HL-34 did not, indicating that the EGFR contains one actin-binding site. These results demonstrate that the EGFR is an actin-binding protein which binds to actin via a domain containing amino acids residues 984-996. PMID:1383230

  15. Crystal structure of a nuclear actin ternary complex.

    PubMed

    Cao, Tingting; Sun, Lingfei; Jiang, Yuxiang; Huang, Shanjin; Wang, Jiawei; Chen, Zhucheng

    2016-08-01

    Actin polymerizes and forms filamentous structures (F-actin) in the cytoplasm of eukaryotic cells. It also exists in the nucleus and regulates various nucleic acid transactions, particularly through its incorporation into multiple chromatin-remodeling complexes. However, the specific structure of actin and the mechanisms that regulate its polymeric nature inside the nucleus remain unknown. Here, we report the crystal structure of nuclear actin (N-actin) complexed with actin-related protein 4 (Arp4) and the helicase-SANT-associated (HSA) domain of the chromatin remodeler Swr1. The inner face and barbed end of N-actin are sequestered by interactions with Arp4 and the HSA domain, respectively, which prevents N-actin from polymerization and binding to many actin regulators. The two major domains of N-actin are more twisted than those of globular actin (G-actin), and its nucleotide-binding pocket is occluded, freeing N-actin from binding to and regulation by ATP. These findings revealed the salient structural features of N-actin that distinguish it from its cytoplasmic counterpart and provide a rational basis for its functions and regulation inside the nucleus. PMID:27457955

  16. The actinome of Dictyostelium discoideum in comparison to actins and actin-related proteins from other organisms.

    PubMed

    Joseph, Jayabalan M; Fey, Petra; Ramalingam, Nagendran; Liu, Xiao I; Rohlfs, Meino; Noegel, Angelika A; Müller-Taubenberger, Annette; Glöckner, Gernot; Schleicher, Michael

    2008-07-09

    Actin belongs to the most abundant proteins in eukaryotic cells which harbor usually many conventional actin isoforms as well as actin-related proteins (Arps). To get an overview over the sometimes confusing multitude of actins and Arps, we analyzed the Dictyostelium discoideum actinome in detail and compared it with the genomes from other model organisms. The D. discoideum actinome comprises 41 actins and actin-related proteins. The genome contains 17 actin genes which most likely arose from consecutive gene duplications, are all active, in some cases developmentally regulated and coding for identical proteins (Act8-group). According to published data, the actin fraction in a D. discoideum cell consists of more than 95% of these Act8-type proteins. The other 16 actin isoforms contain a conventional actin motif profile as well but differ in their protein sequences. Seven actin genes are potential pseudogenes. A homology search of the human genome using the most typical D. discoideum actin (Act8) as query sequence finds the major actin isoforms such as cytoplasmic beta-actin as best hit. This suggests that the Act8-group represents a nearly perfect actin throughout evolution. Interestingly, limited data from D. fasciculatum, a more ancient member among the social amoebae, show different relationships between conventional actins. The Act8-type isoform is most conserved throughout evolution. Modeling of the putative structures suggests that the majority of the actin-related proteins is functionally unrelated to canonical actin. The data suggest that the other actin variants are not necessary for the cytoskeleton itself but rather regulators of its dynamical features or subunits in larger protein complexes.

  17. Impact of Carbon Nanomaterials on Actin Polymerization.

    PubMed

    Dong, Ying; Sun, Haiyan; Li, Xu; Li, Xin; Zhao, Lina

    2016-03-01

    Many nanomaterials have entered people's daily lives and impact the normal process of biological entities consequently. As one kind of the important nanomaterials, carbon based nanomaterials have invoked a lot of concerns from scientific researches because of their unique physicochemical properties. In eukaryotes, actin is the most abundantly distributed protein in both cytoplasm and cell nucleus, and closely controls the cell proliferation and mobility. Recently, many investigations have found some carbon based nanomaterials can affect actin cytoskeleton remarkably, including fullerenes derivatives, carbon nanotubes, graphene and its derivatives. However, these interaction processes are complicated and the underlying mechanism is far from being understood clearly. In this review, we discussed the different mechanisms of carbon nanomaterials impact on actin polymerization into three pathways, as triggering the signaling pathways from carbon nanomaterials outside of cells, increasing the production of reactive oxygen species from carbon nanomaterials inside of cells and direct interaction from carbon nanomaterials inside of cells. As a result, the dimension and size of carbon nanomaterials play a key role in regulation of actin cytoskeleton. Furthermore, we forecasted the possible investigation strategy for meeting the challenges of the future study on this topic. We hope the findings are helpful in understanding the molecular mechanism in carbon nanomaterials regulating actin polymerization, and provide new insight in novel nanomedicine development for inhibition tumor cell migration. PMID:27455649

  18. The Entamoeba histolytica, Arp2/3 Complex Is Recruited to Phagocytic Cups through an Atypical Kinase EhAK1

    PubMed Central

    Babuta, Mrigya; Mansuri, M Shahid; Bhattacharya, Sudha; Bhattacharya, Alok

    2015-01-01

    The parasite Entamoeba histolytica is the etiological agent of amoebiasis and phagocytosis plays a key role in virulence of this organism. Signaling pathways involved in activation of cytoskeletal dynamics required for phagocytosis remain to be elucidated. Phagocytosis is initiated with sequential recruitment of EhC2PK, EhCaBP1, EhCaBP3 and an atypical kinase EhAK1 after particle attachment. Here we show that EhARPC1, an essential subunit of the actin branching complex Arp 2/3 is recruited to the phagocytic initiation sites by EhAK1. Imaging, expression knockdown of different molecules and pull down experiments suggest that EhARPC1 interacts with EhAK1 and that it is required during initiation of phagocytosis and phagosome formation. Moreover, recruitment of EhARPC2 at the phagocytosis initiation by EhAK1 is also observed, indicating that the Arp 2/3 complex is recruited. In conclusion, these results suggests a novel mechanism of recruitment of Arp 2/3 complex during phagocytosis in E. histolytica. PMID:26646565

  19. A new F-actin structure in fungi: actin ring formation around the cell nucleus of Cryptococcus neoformans.

    PubMed

    Kopecká, Marie; Kawamoto, Susumu; Yamaguchi, Masashi

    2013-04-01

    The F-actin cytoskeleton of Cryptococcus neoformans is known to comprise actin cables, cortical patches and cytokinetic ring. Here, we describe a new F-actin structure in fungi, a perinuclear F-actin collar ring around the cell nucleus, by fluorescent microscopic imaging of rhodamine phalloidin-stained F-actin. Perinuclear F-actin rings form in Cryptococcus neoformans treated with the microtubule inhibitor Nocodazole or with the drug solvent dimethyl sulfoxide (DMSO) or grown in yeast extract peptone dextrose (YEPD) medium, but they are absent in cells treated with Latrunculin A. Perinuclear F-actin rings may function as 'funicular cabin' for the cell nucleus, and actin cables as intracellular 'funicular' suspending nucleus in the central position in the cell and moving nucleus along the polarity axis along actin cables.

  20. Structural Transitions of F-Actin:Espin Bundles

    NASA Astrophysics Data System (ADS)

    Purdy, Kirstin; Bartles, James; Wong, Gerard

    2006-03-01

    Espin is an actin bundling protein involved in the formation of the parallel bundles of filamentous actin in hair cell stereocilia. Mutations in espin are implicated in deafness phenotypes in mice and humans. We present measurements of the F-actin structures induced by wild type and by mutated espin obtained via small angle x-ray scattering and fluorescence microscopy. We found that wild type espin induced a paracrystalline hexagonal array of twisted F-actin, whereas the mutated espin only condensed the F-actin into a nematic-like phase. The possibility of coexisting nematic and bundled actin in mixtures containing both mutant and wild type espins was also investigated.

  1. Nuclear actin and lamins in viral infections.

    PubMed

    Cibulka, Jakub; Fraiberk, Martin; Forstova, Jitka

    2012-03-01

    Lamins are the best characterized cytoskeletal components of the cell nucleus that help to maintain the nuclear shape and participate in diverse nuclear processes including replication or transcription. Nuclear actin is now widely accepted to be another cytoskeletal protein present in the nucleus that fulfills important functions in the gene expression. Some viruses replicating in the nucleus evolved the ability to interact with and probably utilize nuclear actin for their replication, e.g., for the assembly and transport of capsids or mRNA export. On the other hand, lamins play a role in the propagation of other viruses since nuclear lamina may represent a barrier for virions entering or escaping the nucleus. This review will summarize the current knowledge about the roles of nuclear actin and lamins in viral infections.

  2. Actin Filament Segmentation Using Dynamic Programming

    PubMed Central

    Li, Hongsheng; Shen, Tian; Huang, Xiaolei

    2011-01-01

    We introduce a novel algorithm for actin filament segmentation in 2D TIRFM image sequences. This problem is difficult because actin filaments dynamically change shapes during their growth, and the TIRFM images are usually noisy. We ask a user to specify the two tips of a filament of interest in the first frame. We then model the segmentation problem in an image sequence as a temporal chain, where its states are tip locations; given candidate tip locations, actin filaments' body points are inferred by a dynamic programming method, which adaptively generates candidate solutions. Combining candidate tip locations and their inferred body points, the temporal chain model is efficiently optimized using another dynamic programming method. Evaluation on noisy TIRFM image sequences demonstrates the accuracy and robustness of this approach. PMID:21761674

  3. Spontaneous actin dynamics in contractile rings

    NASA Astrophysics Data System (ADS)

    Kruse, Karsten; Wollrab, Viktoria; Thiagarajan, Raghavan; Wald, Anne; Riveline, Daniel

    Networks of polymerizing actin filaments are known to be capable to self-organize into a variety of structures. For example, spontaneous actin polymerization waves have been observed in living cells in a number of circumstances, notably, in crawling neutrophils and slime molds. During later stages of cell division, they can also spontaneously form a contractile ring that will eventually cleave the cell into two daughter cells. We present a framework for describing networks of polymerizing actin filaments, where assembly is regulated by various proteins. It can also include the effects of molecular motors. We show that the molecular processes driven by these proteins can generate various structures that have been observed in contractile rings of fission yeast and mammalian cells. We discuss a possible functional role of each of these patterns. The work was supported by Agence Nationale de la Recherche, France, (ANR-10-LABX-0030-INRT) and by Deutsche Forschungsgemeinschaft through SFB1027.

  4. Human Muscle LIM Protein Dimerizes along the Actin Cytoskeleton and Cross-Links Actin Filaments

    PubMed Central

    Hoffmann, Céline; Moreau, Flora; Moes, Michèle; Luthold, Carole; Dieterle, Monika; Goretti, Emeline; Neumann, Katrin; Steinmetz, André

    2014-01-01

    The muscle LIM protein (MLP) is a nucleocytoplasmic shuttling protein playing important roles in the regulation of myocyte remodeling and adaptation to hypertrophic stimuli. Missense mutations in human MLP or its ablation in transgenic mice promotes cardiomyopathy and heart failure. The exact function(s) of MLP in the cytoplasmic compartment and the underlying molecular mechanisms remain largely unknown. Here, we provide evidence that MLP autonomously binds to, stabilizes, and bundles actin filaments (AFs) independently of calcium and pH. Using total internal reflection fluorescence microscopy, we have shown how MLP cross-links actin filaments into both unipolar and mixed-polarity bundles. Quantitative analysis of the actin cytoskeleton configuration confirmed that MLP substantially promotes actin bundling in live myoblasts. In addition, bimolecular fluorescence complementation (BiFC) assays revealed MLP self-association. Remarkably, BiFC complexes mostly localize along actin filament-rich structures, such as stress fibers and sarcomeres, supporting a functional link between MLP self-association and actin cross-linking. Finally, we have demonstrated that MLP self-associates through its N-terminal LIM domain, whereas it binds to AFs through its C-terminal LIM domain. Together our data support that MLP contributes to the maintenance of cardiomyocyte cytoarchitecture by a mechanism involving its self-association and actin filament cross-linking. PMID:24934443

  5. Fatal congenital myopathy with actin filament deposits.

    PubMed

    Bornemann, A; Petersen, M B; Schmalbruch, H

    1996-07-01

    We present the clinical and morphological findings in a case of progressive congenital myopathy. The symptoms present at birth included severe general muscular hypotonia, diffuse muscular atrophy, arthrogryposis, absence of spontaneous movements, and left ventricular hypertrophy. A biopsy specimen taken from the gastrocnemius muscle when the patient was 2 weeks old revealed deposits which consisted of actin filaments as shown by electron microscopy. The infant was occasionally respirator dependent but was mostly able to breathe unassisted. At the age of 5 months he died of respiratory failure. The actin filament deposits may explain the clinical findings.

  6. Actin Age Orchestrates Myosin-5 and Myosin-6 Runlengths

    PubMed Central

    Zimmermann, Dennis; Santos, Alicja; Kovar, David R.; Rock, Ronald S.

    2015-01-01

    Summary Unlike a static and immobile skeleton, the actin cytoskeleton is a highly dynamic network of filamentous actin (F-actin) polymers that continuously turn over. In addition to generating mechanical forces and sensing mechanical deformation, dynamic F-actin networks serve as cellular tracks for myosin motor traffic. However, much of our mechanistic understanding of processive myosins comes from in vitro studies where motility was studied on pre-assembled and artificially stabilized, static F-actin tracks. In this work, we examine the role of actin dynamics in single-molecule myosin motility using assembling F-actin and the two highly processive motors, myosin-5 and myosin-6. These two myosins have distinct functions in the cell and travel in opposite directions along actin filaments [1–3]. Myosin-5 walks towards the barbed ends of F-actin, traveling to sites of actin polymerization at the cell periphery [4]. Myosin-6 walks towards the pointed end of F-actin [5], traveling towards the cell center along older segments of the actin filament. We find that myosin-5 takes 1.3 to 1.5-fold longer runs on ADP•Pi (young) F-actin, while myosin-6 takes 1.7 to 3.6-fold longer runs along ADP (old) F-actin. These results suggest that conformational differences between ADP•Pi and ADP F-actin tailor these myosins to walk farther toward their preferred actin filament end. Taken together, these experiments define a new mechanism by which myosin traffic may sort to different F-actin networks depending on filament age. PMID:26190073

  7. Functional adaptation between yeast actin and its cognate myosin motors.

    PubMed

    Stark, Benjamin C; Wen, Kuo-Kuang; Allingham, John S; Rubenstein, Peter A; Lord, Matthew

    2011-09-01

    We employed budding yeast and skeletal muscle actin to examine the contribution of the actin isoform to myosin motor function. While yeast and muscle actin are highly homologous, they exhibit different charge density at their N termini (a proposed myosin-binding interface). Muscle myosin-II actin-activated ATPase activity is significantly higher with muscle versus yeast actin. Whether this reflects inefficiency in the ability of yeast actin to activate myosin is not known. Here we optimized the isolation of two yeast myosins to assess actin function in a homogenous system. Yeast myosin-II (Myo1p) and myosin-V (Myo2p) accommodate the reduced N-terminal charge density of yeast actin, showing greater activity with yeast over muscle actin. Increasing the number of negative charges at the N terminus of yeast actin from two to four (as in muscle) had little effect on yeast myosin activity, while other substitutions of charged residues at the myosin interface of yeast actin reduced activity. Thus, yeast actin functions most effectively with its native myosins, which in part relies on associations mediated by its outer domain. Compared with yeast myosin-II and myosin-V, muscle myosin-II activity was very sensitive to salt. Collectively, our findings suggest differing degrees of reliance on electrostatic interactions during weak actomyosin binding in yeast versus muscle. Our study also highlights the importance of native actin isoforms when considering the function of myosins. PMID:21757693

  8. Actin nucleators in the nucleus: an emerging theme.

    PubMed

    Weston, Louise; Coutts, Amanda S; La Thangue, Nicholas B

    2012-08-01

    Actin is an integral component of the cytoskeleton, forming a plethora of macromolecular structures that mediate various cellular functions. The formation of such structures relies on the ability of actin monomers to associate into polymers, and this process is regulated by actin nucleation factors. These factors use monomeric actin pools at specific cellular locations, thereby permitting rapid actin filament formation when required. It has now been established that actin is also present in the nucleus, where it is implicated in chromatin remodelling and the regulation of eukaryotic gene transcription. Notably, the presence of typical actin filaments in the nucleus has not been demonstrated directly. However, studies in recent years have provided evidence for the nuclear localisation of actin nucleation factors that promote cytoplasmic actin polymerisation. Their localisation to the nucleus suggests that these proteins mediate collaboration between the cytoskeleton and the nucleus, which might be dependent on their ability to promote actin polymerisation. The nature of this cooperation remains enigmatic and it will be important to elucidate the physiological relevance of the link between cytoskeletal actin networks and nuclear events. This Commentary explores the current evidence for the nuclear roles of actin nucleation factors. Furthermore, the implication of actin-associated proteins in relaying exogenous signals to the nucleus, particularly in response to cellular stress, will be considered.

  9. Dynamic Regulation of Sarcomeric Actin Filaments in Striated Muscle

    PubMed Central

    Ono, Shoichiro

    2010-01-01

    In striated muscle, the actin cytoskeleton is differentiated into myofibrils. Actin and myosin filaments are organized in sarcomeres and specialized for producing contractile forces. Regular arrangement of actin filaments with uniform length and polarity is critical for the contractile function. However, the mechanisms of assembly and maintenance of sarcomeric actin filaments in striated muscle are not completely understood. Live imaging of actin in striated muscle has revealed that actin subunits within sarcomeric actin filaments are dynamically exchanged without altering overall sarcomeric structures. A number of regulators for actin dynamics have been identified, and malfunction of these regulators often result in disorganization of myofibril structures or muscle diseases. Therefore, proper regulation of actin dynamics in striated muscle is critical for assembly and maintenance of functional myofibrils. Recent studies have suggested that both enhancers of actin dynamics and stabilizers of actin filaments are important for sarcomeric actin organization. Further investigation of the regulatory mechanism of actin dynamics in striated muscle should be a key to understanding how myofibrils develop and operate. © 2010 Wiley-Liss, Inc. PMID:20737540

  10. Actin3 promoter reveals undulating F-actin bundles at shanks and dynamic F-actin meshworks at tips of tip-growing pollen tubes.

    PubMed

    Jásik, Ján; Mičieta, Karol; Siao, Wei; Voigt, Boris; Stuchlík, Stanislav; Schmelzer, Elmon; Turňa, Ján; Baluška, František

    2016-01-01

    The dynamic actin cytoskeleton of pollen tubes is both the driver of the tip growth and the organizer of cell polarity. In order to understand this fast re-arranging cytoskeletal system, we need reliable constructs expressed under relevant promoters. Here we are reporting that the Lifeact reporter, expressed under the pollen-specific Actin3 promoter, visualizes very dynamic F-actin elements both in germinating pollen grains and tip-growing pollen tubes. Importantly, we have documented very active actin polymerization at the cell periphery, especially in the bulging area during pollen germination and in the apical clear zone. Expression of the Lifeact reporter under control of the pollen-specific Actin3 promoter revealed 2 new aspects: (i) long F-actin bundles in pollen tube shanks are dynamic, showing undulating movements, (ii) subapical 'actin collars' or 'fringes' are absent.

  11. Actin3 promoter reveals undulating F-actin bundles at shanks and dynamic F-actin meshworks at tips of tip-growing pollen tubes

    PubMed Central

    Jásik, Ján; Mičieta, Karol; Siao, Wei; Voigt, Boris; Stuchlík, Stanislav; Schmelzer, Elmon; Turňa, Ján; Baluška, František

    2016-01-01

    ABSTRACT The dynamic actin cytoskeleton of pollen tubes is both the driver of the tip growth and the organizer of cell polarity. In order to understand this fast re-arranging cytoskeletal system, we need reliable constructs expressed under relevant promoters. Here we are reporting that the Lifeact reporter, expressed under the pollen-specific Actin3 promoter, visualizes very dynamic F-actin elements both in germinating pollen grains and tip-growing pollen tubes. Importantly, we have documented very active actin polymerization at the cell periphery, especially in the bulging area during pollen germination and in the apical clear zone. Expression of the Lifeact reporter under control of the pollen-specific Actin3 promoter revealed 2 new aspects: (i) long F-actin bundles in pollen tube shanks are dynamic, showing undulating movements, (ii) subapical ‘actin collars’ or ‘fringes’ are absent. PMID:26980067

  12. [Cytoskeletal actin and its associated proteins. Some examples in Protista].

    PubMed

    Guillén, N; Carlier, M F; Brugerolle, G; Tardieux, I; Ausseil, J

    1998-06-01

    Many processes, cell motility being an example, require cells to remodel the actin cytoskeleton in response to both intracellular and extracellular signals. Reorganization of the actin cytoskeleton involves the rapid disassembly and reassembly of actin filaments, a phenomenon regulated by the action of particular actin-binding proteins. In recent years, an interest in studying actin regulation in unicellular organisms has arisen. Parasitic protozoan are among these organisms and studies of the cytoskeleton functions of these protozoan are relevant related to either cell biology or pathogenicity. To discuss recent data in this field, a symposium concerning "Actin and actin-binding proteins in protists" was held on May 8-11 in Paris, France, during the XXXV meeting of the French Society of Protistology. As a brief summary of the symposium we report here findings concerning the in vitro actin dynamic assembly, as well as the characterization of several actin-binding proteins from the parasitic protozoan Entamoeba histolytica, Trichomonas vaginalis and Plasmodium knowlesi. In addition, localization of actin in non-pathogen protists such as Prorocentrum micans and Crypthecodinium cohnii is also presented. The data show that some actin-binding proteins facilitate organization of filaments into higher order structures as pseudopods, while others have regulatory functions, indicating very particular roles for actin-binding proteins. One of the proteins discussed during the symposium, the actin depolymerizing factor ADF, was shown to enhance the treadmilling rate of actin filaments. In vitro, ADF binds to the ADP-bound forms of G-actin and F-actin, thereby participating in and changing the rate of actin assembly. Biochemical approaches allowed the identification of a protein complex formed by HSP/C70-cap32-34 which might also be involved in depolymerization of F-actin in P. knowlesi. Molecular and cellular approaches were used to identify proteins such as ABP-120 and myosin

  13. The actin cytoskeleton as a sensor and mediator of apoptosis

    PubMed Central

    Desouza, Melissa; Gunning, Peter W.; Stehn, Justine R.

    2012-01-01

    Apoptosis is an important biological process required for the removal of unwanted or damaged cells. Mounting evidence implicates the actin cytoskeleton as both a sensor and mediator of apoptosis. Studies also suggest that actin binding proteins (ABPs) significantly contribute to apoptosis and that actin dynamics play a key role in regulating apoptosis signaling. Changes in the organization of the actin cytoskeleton has been attributed to the process of malignant transformation and it is hypothesized that remodeling of the actin cytoskeleton may enable tumor cells to evade normal apoptotic signaling. This review aims to illuminate the role of the actin cytoskeleton in apoptosis by systematically analyzing how actin and ABPs regulate different apoptosis pathways and to also highlight the potential for developing novel compounds that target tumor-specific actin filaments. PMID:22880146

  14. CNS myelin wrapping is driven by actin disassembly.

    PubMed

    Zuchero, J Bradley; Fu, Meng-Meng; Sloan, Steven A; Ibrahim, Adiljan; Olson, Andrew; Zaremba, Anita; Dugas, Jason C; Wienbar, Sophia; Caprariello, Andrew V; Kantor, Christopher; Leonoudakis, Dmitri; Leonoudakus, Dmitri; Lariosa-Willingham, Karen; Kronenberg, Golo; Gertz, Karen; Soderling, Scott H; Miller, Robert H; Barres, Ben A

    2015-07-27

    Myelin is essential in vertebrates for the rapid propagation of action potentials, but the molecular mechanisms driving its formation remain largely unknown. Here we show that the initial stage of process extension and axon ensheathment by oligodendrocytes requires dynamic actin filament assembly by the Arp2/3 complex. Unexpectedly, subsequent myelin wrapping coincides with the upregulation of actin disassembly proteins and rapid disassembly of the oligodendrocyte actin cytoskeleton and does not require Arp2/3. Inducing loss of actin filaments drives oligodendrocyte membrane spreading and myelin wrapping in vivo, and the actin disassembly factor gelsolin is required for normal wrapping. We show that myelin basic protein, a protein essential for CNS myelin wrapping whose role has been unclear, is required for actin disassembly, and its loss phenocopies loss of actin disassembly proteins. Together, these findings provide insight into the molecular mechanism of myelin wrapping and identify it as an actin-independent form of mammalian cell motility.

  15. Actin network architecture can determine myosin motor activity.

    PubMed

    Reymann, Anne-Cécile; Boujemaa-Paterski, Rajaa; Martiel, Jean-Louis; Guérin, Christophe; Cao, Wenxiang; Chin, Harvey F; De La Cruz, Enrique M; Théry, Manuel; Blanchoin, Laurent

    2012-06-01

    The organization of actin filaments into higher-ordered structures governs eukaryotic cell shape and movement. Global actin network size and architecture are maintained in a dynamic steady state through regulated assembly and disassembly. Here, we used experimentally defined actin structures in vitro to investigate how the activity of myosin motors depends on network architecture. Direct visualization of filaments revealed myosin-induced actin network deformation. During this reorganization, myosins selectively contracted and disassembled antiparallel actin structures, while parallel actin bundles remained unaffected. The local distribution of nucleation sites and the resulting orientation of actin filaments appeared to regulate the scalability of the contraction process. This "orientation selection" mechanism for selective contraction and disassembly suggests how the dynamics of the cellular actin cytoskeleton can be spatially controlled by actomyosin contractility.

  16. Utilization of paramagnetic relaxation enhancements for structural analysis of actin-binding proteins in complex with actin

    PubMed Central

    Huang, Shuxian; Umemoto, Ryo; Tamura, Yuki; Kofuku, Yutaka; Uyeda, Taro Q. P.; Nishida, Noritaka; Shimada, Ichio

    2016-01-01

    Actin cytoskeleton dynamics are controlled by various actin binding proteins (ABPs) that modulate the polymerization of the monomeric G-actin and the depolymerization of filamentous F-actin. Although revealing the structures of the actin/ABP complexes is crucial to understand how the ABPs regulate actin dynamics, the X-ray crystallography and cryoEM methods are inadequate to apply for the ABPs that interact with G- or F-actin with lower affinity or multiple binding modes. In this study, we aimed to establish the alternative method to build a structural model of G-actin/ABP complexes, utilizing the paramagnetic relaxation enhancement (PRE) experiments. Thymosin β4 (Tβ4) was used as a test case for validation, since its structure in complex with G-actin was reported recently. Recombinantly expressed G-actin, containing a cysteine mutation, was conjugated with a nitroxyl spin label at the specific site. Based on the intensity ratio of the 1H-15N HSQC spectra of Tβ4 in the complex with G-actin in the paramagnetic and diamagnetic states, the distances between the amide groups of Tβ4 and the spin label of G-actin were estimated. Using the PRE-derived distance constraints, we were able to compute a well-converged docking structure of the G-actin/Tβ4 complex that shows great accordance with the reference structure. PMID:27654858

  17. 77 FR 16314 - Alaska Disaster #AK-00024

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-03-20

    ... ADMINISTRATION Alaska Disaster AK-00024 AGENCY: U.S. Small Business Administration. ACTION: Notice. SUMMARY: This is a notice of an Administrative declaration of a disaster for the State of Alaska dated 03/13/2012... INFORMATION CONTACT: A. Escobar, Office of Disaster Assistance, U.S. Small Business Administration, 409...

  18. 78 FR 39822 - Alaska Disaster #AK-00028

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-07-02

    ... ADMINISTRATION Alaska Disaster AK-00028 AGENCY: U.S. Small Business Administration. ACTION: Notice SUMMARY: This is a Notice of the Presidential declaration of a major disaster for the State of Alaska (FEMA-4122-DR... INFORMATION CONTACT: A. Escobar, Office of Disaster Assistance, U.S. Small Business Administration, 409...

  19. 78 FR 39821 - Alaska Disaster #AK-00029

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-07-02

    ... ADMINISTRATION Alaska Disaster AK-00029 AGENCY: U.S. Small Business Administration. ACTION: Notice. SUMMARY: This is a Notice of the Presidential declaration of a major disaster for Public Assistance Only for the..., Fort Worth, TX 76155. FOR FURTHER INFORMATION CONTACT: A. Escobar, Office of Disaster Assistance,...

  20. 76 FR 35936 - Alaska Disaster #AK-00020

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-06-20

    ... From the Federal Register Online via the Government Publishing Office U.S. SMALL BUSINESS ADMINISTRATION Alaska Disaster AK-00020 AGENCY: U.S. Small Business Administration. ACTION: Notice. SUMMARY: This... completed loan applications to: U.S. Small Business Administration, Processing and Disbursement...

  1. Interaction of profilin with the barbed end of actin filaments.

    PubMed

    Courtemanche, Naomi; Pollard, Thomas D

    2013-09-17

    Profilin binds not only to actin monomers but also to the barbed end of the actin filament, where it inhibits association of subunits. To address open questions about the interactions of profilin with barbed ends, we measured the effects of a wide range of concentrations of Homo sapiens profilin 1 on the rate of elongation of individual skeletal muscle actin filaments by total internal reflection fluorescence microscopy. Much higher concentrations of profilin were required to stop elongation by AMP-PNP-actin monomers than ADP-actin monomers. High concentrations of profilin depolymerized barbed ends at a rate much faster than the spontaneous dissociation rates of Mg-ATP-, Mg-AMP-PNP-, Mg-ADP-Pi-, and Mg-ADP-actin subunits. Fitting a thermodynamic model to these data allowed us to determine the affinities of profilin and profilin-actin for barbed ends and the influence of the nucleotide bound to actin on these interactions. Profilin has a much higher affinity for ADP-actin filament barbed ends (Kd = 1 μM) than AMP-PNP-actin filament barbed ends (Kd = 226 μM). ADP-actin monomers associated with profilin bind to ADP-actin filament barbed ends 10% as fast as free ADP-actin monomers, but bound profilin does not affect the rate of association of AMP-PNP-actin monomers with barbed ends. The differences in the affinities of AMP-PNP- and ADP-bound barbed ends for profilin and profilin-actin suggest that conformations of barbed end subunits differ from those of monomers and change upon nucleotide hydrolysis and phosphate release. A structural model revealed minor steric clashes between profilin and actin subunits at the barbed end that explain the biochemical results.

  2. Actin of Beta vulgaris seedlings under the clinorotation

    NASA Astrophysics Data System (ADS)

    Kozeko, L. Ye.

    We study the influence of altered gravity on actin expression in roots of Beta vulguris seedlings grown on the horizontal clinostat (2 rpm) from seed germination for three days. It is shown that the total actin quantity was not influenced. Three actin isoforms are revealed; a relative protein quantity of these isoforms was similar both in clinorotated seedlings and in ones grown in norm. This point to stable expression of actin under the altered gravity conditions.

  3. Curvature and torsion in growing actin networks

    NASA Astrophysics Data System (ADS)

    Shaevitz, Joshua W.; Fletcher, Daniel A.

    2008-06-01

    Intracellular pathogens such as Listeria monocytogenes and Rickettsia rickettsii move within a host cell by polymerizing a comet-tail of actin fibers that ultimately pushes the cell forward. This dense network of cross-linked actin polymers typically exhibits a striking curvature that causes bacteria to move in gently looping paths. Theoretically, tail curvature has been linked to details of motility by considering force and torque balances from a finite number of polymerizing filaments. Here we track beads coated with a prokaryotic activator of actin polymerization in three dimensions to directly quantify the curvature and torsion of bead motility paths. We find that bead paths are more likely to have low rather than high curvature at any given time. Furthermore, path curvature changes very slowly in time, with an autocorrelation decay time of 200 s. Paths with a small radius of curvature, therefore, remain so for an extended period resulting in loops when confined to two dimensions. When allowed to explore a three-dimensional (3D) space, path loops are less evident. Finally, we quantify the torsion in the bead paths and show that beads do not exhibit a significant left- or right-handed bias to their motion in 3D. These results suggest that paths of actin-propelled objects may be attributed to slow changes in curvature, possibly associated with filament debranching, rather than a fixed torque.

  4. Viscoelastic properties of actin-coated membranes

    NASA Astrophysics Data System (ADS)

    Helfer, E.; Harlepp, S.; Bourdieu, L.; Robert, J.; Mackintosh, F. C.; Chatenay, D.

    2001-02-01

    In living cells, cytoskeletal filaments interact with the plasma membrane to form structures that play a key role in cell shape and mechanical properties. To study the interaction between these basic components, we designed an in vitro self-assembled network of actin filaments attached to the outer surface of giant unilamellar vesicles. Optical tweezers and single-particle tracking experiments are used to study the rich dynamics of these actin-coated membranes (ACM). We show that microrheology studies can be carried out on such an individual microscopic object. The principle of the experiment consists in measuring the thermally excited position fluctuations of a probe bead attached biochemically to the membrane. We propose a model that relates the power spectrum of these thermal fluctuations to the viscoelastic properties of the membrane. The presence of the actin network modifies strongly the membrane dynamics with respect to a fluid, lipid bilayer one. It induces first a finite (ω=0) two-dimensional (2D) shear modulus G02D~0.5 to 5 μN/m in the membrane plane. Moreover, the frequency dependence at high frequency of the shear modulus [G'2D(f )~f0.85+/-0.07] and of the bending modulus (κACM(f)~f0.55+/-0.21) demonstrate the viscoelastic behavior of the composite membrane. These results are consistent with a common exponent of 0.75 for both moduli as expected from our model and from prior measurements on actin solutions.

  5. Endothelial actin-binding proteins and actin dynamics in leukocyte transendothelial migration.

    PubMed

    Schnoor, Michael

    2015-04-15

    The endothelium is the first barrier that leukocytes have to overcome during recruitment to sites of inflamed tissues. The leukocyte extravasation cascade is a complex multistep process that requires the activation of various adhesion molecules and signaling pathways, as well as actin remodeling, in both leukocytes and endothelial cells. Endothelial adhesion molecules, such as E-selectin or ICAM-1, are connected to the actin cytoskeleton via actin-binding proteins (ABPs). Although the contribution of receptor-ligand interactions to leukocyte extravasation has been studied extensively, the contribution of endothelial ABPs to the regulation of leukocyte adhesion and transendothelial migration remains poorly understood. This review focuses on recently published evidence that endothelial ABPs, such as cortactin, myosin, or α-actinin, regulate leukocyte extravasation by controlling actin dynamics, biomechanical properties of endothelia, and signaling pathways, such as GTPase activation, during inflammation. Thus, ABPs may serve as targets for novel treatment strategies for disorders characterized by excessive leukocyte recruitment.

  6. Competition of two distinct actin networks for actin defines a bistable switch for cell polarization

    PubMed Central

    Lomakin, Alexis J.; Lee, Kun-Chun; Han, Sangyoon J.; Bui, D A.; Davidson, Michael; Mogilner, Alex; Danuser, Gaudenz

    2015-01-01

    Symmetry-breaking polarization enables functional plasticity of cells and tissues and is yet not well understood. Here we show that epithelial cells, hard-wired to maintain a static morphology and to preserve tissue organization, can spontaneously switch to a migratory polarized phenotype upon relaxation of the actomyosin cytoskeleton. We find that myosin-II engages actin in the formation of cortical actomyosin bundles and thus makes it unavailable for deployment in the process of dendritic growth normally driving cell motility. At low contractility regimes epithelial cells polarize in a front-back manner due to emergence of actin retrograde flows powered by dendritic polymerization of actin. Coupled to cell movement, the flows transport myosin-II from the front to the back of the cell, where the motor locally “locks” actin in contractile bundles. This polarization mechanism could be employed by embryonic and cancer epithelial cells in microenvironments where high contractility-driven cell motion is inefficient. PMID:26414403

  7. Actin cytoskeleton demonstration in Trichomonas vaginalis and in other trichomonads.

    PubMed

    Brugerolle, G; Bricheux, G; Coffe, G

    1996-01-01

    The flagellate form of Trichomonas vaginalis (T v) transforms to amoeboid cells upon adherence to converslips. They grow and their nuclei divide without undergoing cytokinesis, yielding giant cells and a monolayer of T v F-actin was demonstrated in Trichomonas vaginalis by fluorescence microscopy using phalloidin and an anti-actin mAb which labelled the cytoplasm of both the flagellate and amoeboid forms. Comparative electrophoresis and immunoblotting established that the actin band has the same 42 kDa as muscle actin, but 2-D electrophoresis resolved the actin band into four spots; the two major spots observed were superimposable with major muscle actin isoforms. Electron microscopy demonstrated an ectoplasmic microfibrillar layer along the adhesion zone of amoeboid T v adhering to coverslips. Immunogold staining, using anti-actin monoclonal antibodies demonstrated that this layer was mainly composed of actin microfilaments. A comparative immunoblotting study comprising seven trichomonad species showed that all trichomonads studied expressed actin. The mAb Sigma A-4700 specific for an epitope on the actin C-terminal sequence labelled only actin of Trichomonas vaginalis, Tetratrichomonas gallinarum. Trichomitus batrachorum and Hypotrichomonas acosta, but not the actin of Tritrichomonas foetus, Tritrichomonas augusta and Monocercomonas sp. This discrimination between a 'trichomonas branch' and a 'tritrichomonas branch' is congruent with inferred sequence phylogeny from SSu rRNA and with classical phylogeny of trichomonads. PMID:9175265

  8. Actin-organising properties of the muscular dystrophy protein myotilin.

    PubMed

    von Nandelstadh, Pernilla; Grönholm, Mikaela; Moza, Monica; Lamberg, Arja; Savilahti, Harri; Carpén, Olli

    2005-10-15

    Myotilin is a sarcomeric Z-disc protein that binds F-actin directly and bundles actin filaments, although it does not contain a conventional actin-binding domain. Expression of mutant myotilin leads to sarcomeric alterations in the dominantly inherited limb-girdle muscular dystrophy 1A and in myofibrillar myopathy/desmin-related myopathy. Together, with previous in vitro studies, this indicates that myotilin has an important function in the assembly and maintenance of Z-discs. This study characterises further the interaction between myotilin and actin. Functionally important regions in myotilin were identified by actin pull-down and yeast two-hybrid assays and with a novel strategy that combines in vitro DNA transposition-based peptide insertion mutagenesis with phenotype analysis in yeast cells. The shortest fragment to bind actin was the second Ig domain together with a short C-terminal sequence. Concerted action of the first and second Ig domain was, however, necessary for the functional activity of myotilin, as verified by analysis of transposon mutants, actin binding and phenotypic effect in mammalian cells. Furthermore, the Ig domains flanked with N- and C-terminal regions were needed for actin-bundling, indicating that the mere actin-binding sequence was insufficient for the actin-regulating activity. None of the four known disease-associated mutations altered the actin-organising ability. These results, together with previous studies in titin and kettin, identify the Ig domain as an actin-binding unit.

  9. Prostaglandins temporally regulate cytoplasmic actin bundle formation during Drosophila oogenesis.

    PubMed

    Spracklen, Andrew J; Kelpsch, Daniel J; Chen, Xiang; Spracklen, Cassandra N; Tootle, Tina L

    2014-02-01

    Prostaglandins (PGs)--lipid signals produced downstream of cyclooxygenase (COX) enzymes--regulate actin dynamics in cell culture and platelets, but their roles during development are largely unknown. Here we define a new role for Pxt, the Drosophila COX-like enzyme, in regulating the actin cytoskeleton--temporal restriction of actin remodeling during oogenesis. PGs are required for actin filament bundle formation during stage 10B (S10B). In addition, loss of Pxt results in extensive early actin remodeling, including actin filaments and aggregates, within the posterior nurse cells of S9 follicles; wild-type follicles exhibit similar structures at a low frequency. Hu li tai shao (Hts-RC) and Villin (Quail), an actin bundler, localize to all early actin structures, whereas Enabled (Ena), an actin elongation factor, preferentially localizes to those in pxt mutants. Reduced Ena levels strongly suppress early actin remodeling in pxt mutants. Furthermore, loss of Pxt results in reduced Ena localization to the sites of bundle formation during S10B. Together these data lead to a model in which PGs temporally regulate actin remodeling during Drosophila oogenesis by controlling Ena localization/activity, such that in S9, PG signaling inhibits, whereas at S10B, it promotes Ena-dependent actin remodeling.

  10. Shape control of lipid bilayer membranes by confined actin bundles.

    PubMed

    Tsai, Feng-Ching; Koenderink, Gijsje Hendrika

    2015-12-01

    In living cells, lipid membranes and biopolymers determine each other's conformation in a delicate force balance. Cellular polymers such as actin filaments are strongly confined by the plasma membrane in cell protrusions such as lamellipodia and filopodia. Conversely, protrusion formation is facilitated by actin-driven membrane deformation and these protrusions are maintained by dense actin networks or bundles of actin filaments. Here we investigate the mechanical interplay between actin bundles and lipid bilayer membranes by reconstituting a minimal model system based on cell-sized liposomes with encapsulated actin filaments bundled by fascin. To address the competition between the deformability of the membrane and the enclosed actin bundles, we tune the bundle stiffness (through the fascin-to-actin molar ratio) and the membrane rigidity (through protein decoration). Using confocal microscopy and quantitative image analysis, we show that actin bundles deform the liposomes into a rich set of morphologies. For liposomes having a small membrane bending rigidity, the actin bundles tend to generate finger-like membrane protrusions that resemble cellular filopodia. Stiffer bundles formed at high crosslink density stay straight in the liposome body, whereas softer bundles formed at low crosslink density are bent and kinked. When the membrane has a large bending rigidity, membrane protrusions are suppressed. In this case, membrane enclosure forces the actin bundles to organize into cortical rings, to minimize the energy cost associated with filament bending. Our results highlight the importance of taking into account mechanical interactions between the actin cytoskeleton and the membrane to understand cell shape control.

  11. Unconventional actins and actin-binding proteins in human protozoan parasites.

    PubMed

    Gupta, C M; Thiyagarajan, S; Sahasrabuddhe, A A

    2015-06-01

    Actin and its regulatory proteins play a key role in several essential cellular processes such as cell movement, intracellular trafficking and cytokinesis in most eukaryotes. While these proteins are highly conserved in higher eukaryotes, a number of unicellular eukaryotic organisms contain divergent forms of these proteins which have highly unusual biochemical and structural properties. Here, we review the biochemical and structural properties of these unconventional actins and their core binding proteins which are present in commonly occurring human protozoan parasites.

  12. FMNL3 FH2-actin structure gives insight into formin-mediated actin nucleation and elongation

    SciTech Connect

    Thompson, Morgan E; Heimsath, Ernest G; Gauvin, Timothy J; Higgs, Henry N; Kull, F Jon

    2012-12-09

    Formins are actin-assembly factors that act in a variety of actin-based processes. The conserved formin homology 2 (FH2) domain promotes filament nucleation and influences elongation through interaction with the barbed end. FMNL3 is a formin that induces assembly of filopodia but whose FH2 domain is a poor nucleator. The 3.4-Å structure of a mouse FMNL3 FH2 dimer in complex with tetramethylrhodamine-actin uncovers details of formin-regulated actin elongation. We observe distinct FH2 actin-binding regions; interactions in the knob and coiled-coil subdomains are necessary for actin binding, whereas those in the lasso-post interface are important for the stepping mechanism. Biochemical and cellular experiments test the importance of individual residues for function. This structure provides details for FH2-mediated filament elongation by processive capping and supports a model in which C-terminal non-FH2 residues of FMNL3 are required to stabilize the filament nucleus.

  13. Structure of a Bud6/actin complex reveals a novel WH2-like actin monomer recruitment motif

    PubMed Central

    Park, Eunyoung; Graziano, Brian R.; Zheng, Wei; Garabedian, Mikael; Goode, Bruce L.; Eck, Michael J.

    2015-01-01

    SUMMARY In budding yeast, the actin-binding protein Bud6 cooperates with formins Bni1 and Bnr1 to catalyze the assembly of actin filaments. The nucleation-enhancing activity of Bud6 requires both a “core” domain that binds to the formin and a “flank” domain that binds monomeric actin. Here we describe the structure of the Bud6 flank domain in complex with actin. Two helices in Bud6flank interact with actin; one binds in a groove at the barbed-end of the actin monomer in a manner closely resembling the helix of WH2 domains, a motif found in many actin nucleation factors. The second helix rises along the face of actin. Mutational analysis verifies the importance of these Bud6-actin contacts for nucleation-enhancing activity. The Bud6 binding site on actin overlaps with that of the formin FH2 domain and is also incompatible with inter-subunit contacts in F-actin, suggesting that Bud6 interacts only transiently with actin monomers during filament nucleation. PMID:26118535

  14. Structure of a Bud6/Actin Complex Reveals a Novel WH2-like Actin Monomer Recruitment Motif.

    PubMed

    Park, Eunyoung; Graziano, Brian R; Zheng, Wei; Garabedian, Mikael; Goode, Bruce L; Eck, Michael J

    2015-08-01

    In budding yeast, the actin-binding protein Bud6 cooperates with formins Bni1 and Bnr1 to catalyze the assembly of actin filaments. The nucleation-enhancing activity of Bud6 requires both a "core" domain that binds to the formin and a "flank" domain that binds monomeric actin. Here, we describe the structure of the Bud6 flank domain in complex with actin. Two helices in Bud6(flank) interact with actin; one binds in a groove at the barbed end of the actin monomer in a manner closely resembling the helix of WH2 domains, a motif found in many actin nucleation factors. The second helix rises along the face of actin. Mutational analysis verifies the importance of these Bud6-actin contacts for nucleation-enhancing activity. The Bud6 binding site on actin overlaps with that of the formin FH2 domain and is also incompatible with inter-subunit contacts in F-actin, suggesting that Bud6 interacts only transiently with actin monomers during filament nucleation.

  15. The origin and evolution of green algal and plant actins.

    PubMed

    An, S S; Möpps, B; Weber, K; Bhattacharya, D

    1999-02-01

    The Viridiplantae are subdivided into two groups: the Chlorophyta, which includes the Chlorophyceae, Trebouxiophyceae, Ulvophyceae, and Prasinophyceae; and the Streptophyta, which includes the Charophyceae and all land plants. Within the Streptophyta, the actin genes of the angiosperms diverge nearly simultaneously from each other before the separation of monocots and dicots. Previous evolutionary analyses have provided limited insights into the gene duplications that have produced these complex gene families. We address the origin and diversification of land plant actin genes by studying the phylogeny of actins within the green algae, ferns, and fern allies. Partial genomic sequences or cDNAs encoding actin were characterized from Cosmarium botrytis (Zygnematales), Selaginella apoda (Selaginellales), Anemia phyllitidis (Polypodiales), and Psilotum triquetrum (Psilotales). Selaginella contains at least two actin genes. One sequence (Ac2) diverges within a group of fern sequences that also includes the Psilotum Ac1 actin gene and one gymnosperm sequence (Cycas revoluta Cyc3). This clade is positioned outside of the angiosperm actin gene radiation. The second Selaginella sequence (Ac1) is the sister to all remaining land plant actin sequences, although the internal branches in this portion of the tree are very short. Use of complete actin-coding regions in phylogenetic analyses provides support for the separation of angiosperm actins into two classes. N-terminal "signature" sequence analyses support these groupings. One class (VEG) includes actin genes that are often expressed in vegetative structures. The second class (REP) includes actin genes that trace their ancestry within the vegetative actins and contains members that are largely expressed in reproductive structures. Analysis of intron positions within actin genes shows that sequences from both Selaginella and Cosmarium contain the conserved 20-3, 152-1, and 356-3 introns found in many members of the

  16. Demonstration of prominent actin filaments in the root columella.

    PubMed

    Collings, D A; Zsuppan, G; Allen, N S; Blancaflor, E B

    2001-02-01

    The distribution of actin filaments within the gravity-sensing columella cells of plant roots remains poorly understood, with studies over numerous years providing inconsistent descriptions of actin organization in these cells. This uncertainty in actin organization, and thus in actin's role in graviperception and gravisignaling, has led us to investigate actin arrangements in the columella cells of Zea mays L., Medicago truncatula Gaertn., Linum usitatissiilium L. and Nicotianla benthamiana Domin. Actin organization was examined using a combination of optimized immunofluorescence techniques, and an improved fluorochrome-conjugated phalloidin labeling method reliant on 3-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS) cross-linking combined with glycerol permeabilization. Confocal microscopy of root sections labeled with anti-actin antibodies revealed patterns suggestive of actin throughout the columella region. These patterns included short and fragmented actin bundles, fluorescent rings around amyloplasts and intense fluorescence originating from the nucleus. Additionally, confocal microscopy of MBS-stabilized and Alexa Fluor-phalloidin-labeled root sections revealed a previously undetected state of actin organization in the columella. Discrete actin structures surrounded the amyloplasts and prominent actin cables radiated from the nuclear surface toward the cell periphery. Furthermore, the cortex of the columella cells contained fine actin bundles (or single filaments) that had a predominant transverse orientation. We also used confocal microscopy of plant roots expressing endoplasmic reticulum (ER)-targeted green fluorescent protein to demonstrate rapid ER movements within the columella cells, suggesting that the imaged actin network is functional. The successful identification of discrete actin structures in the root columella cells forms the perception and signaling. PMID:11289604

  17. Exploring the Stability Limits of Actin and Its Suprastructures

    PubMed Central

    Rosin, Christopher; Erlkamp, Mirko; Ecken, Julian von der; Raunser, Stefan; Winter, Roland

    2014-01-01

    Actin is the main component of the microfilament system in eukaryotic cells and can be found in distinct morphological states. Global (G)-actin is able to assemble into highly organized, supramolecular cellular structures known as filamentous (F)-actin and bundled (B)-actin. To evaluate the structure and stability of G-, F-, and B-actin over a wide range of temperatures and pressures, we used Fourier transform infrared spectroscopy in combination with differential scanning and pressure perturbation calorimetry, small-angle x-ray scattering, laser confocal scanning microscopy, and transmission electron microscopy. Our analysis was designed to provide new (to our knowledge) insights into the stabilizing forces of actin self-assembly and to reveal the stability of the actin polymorphs, including in conditions encountered in extreme environments. In addition, we sought to explain the limited pressure stability of actin self-assembly observed in vivo. G-actin is not only the least temperature-stable but also the least pressure-stable actin species. Under abyssal conditions, where temperatures as low as 1–4°C and pressures up to 1 kbar are reached, G-actin is hardly stable. However, the supramolecular assemblies of actin are stable enough to withstand the extreme conditions usually encountered on Earth. Beyond ∼3–4 kbar, filamentous structures disassemble, and beyond ∼4 kbar, complete dissociation of F-actin structures is observed. Between ∼1 and 2 kbar, some disordering of actin assemblies commences, in agreement with in vivo observations. The limited pressure stability of the monomeric building block seems to be responsible for the suppression of actin assembly in the kbar pressure range. PMID:25517163

  18. Demonstration of prominent actin filaments in the root columella

    NASA Technical Reports Server (NTRS)

    Collings, D. A.; Zsuppan, G.; Allen, N. S.; Blancaflor, E. B.; Brown, C. S. (Principal Investigator)

    2001-01-01

    The distribution of actin filaments within the gravity-sensing columella cells of plant roots remains poorly understood, with studies over numerous years providing inconsistent descriptions of actin organization in these cells. This uncertainty in actin organization, and thus in actin's role in graviperception and gravisignaling, has led us to investigate actin arrangements in the columella cells of Zea mays L., Medicago truncatula Gaertn., Linum usitatissiilium L. and Nicotianla benthamiana Domin. Actin organization was examined using a combination of optimized immunofluorescence techniques, and an improved fluorochrome-conjugated phalloidin labeling method reliant on 3-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS) cross-linking combined with glycerol permeabilization. Confocal microscopy of root sections labeled with anti-actin antibodies revealed patterns suggestive of actin throughout the columella region. These patterns included short and fragmented actin bundles, fluorescent rings around amyloplasts and intense fluorescence originating from the nucleus. Additionally, confocal microscopy of MBS-stabilized and Alexa Fluor-phalloidin-labeled root sections revealed a previously undetected state of actin organization in the columella. Discrete actin structures surrounded the amyloplasts and prominent actin cables radiated from the nuclear surface toward the cell periphery. Furthermore, the cortex of the columella cells contained fine actin bundles (or single filaments) that had a predominant transverse orientation. We also used confocal microscopy of plant roots expressing endoplasmic reticulum (ER)-targeted green fluorescent protein to demonstrate rapid ER movements within the columella cells, suggesting that the imaged actin network is functional. The successful identification of discrete actin structures in the root columella cells forms the perception and signaling.

  19. Actin nucleation and elongation factors: mechanisms and interplay.

    PubMed

    Chesarone, Melissa A; Goode, Bruce L

    2009-02-01

    Cells require actin nucleators to catalyze the de novo assembly of filaments and actin elongation factors to control the rate and extent of polymerization. Nucleation and elongation factors identified to date include Arp2/3 complex, formins, Ena/VASP, and newcomers Spire, Cobl, and Lmod. Here, we discuss recent advances in understanding their activities and mechanisms and new evidence for their cooperation and interaction in vivo. Earlier models had suggested that different nucleators function independently to assemble distinct actin arrays. However, more recent observations indicate that the construction of most cellular actin networks depends on the activities of multiple actin assembly-promoting factors working in concert.

  20. Direct Observation of Tropomyosin Binding to Actin Filaments

    PubMed Central

    Schmidt, William M.; Lehman, William; Moore, Jeffrey R.

    2015-01-01

    Tropomyosin is an elongated α-helical coiled-coil that binds to seven consecutive actin subunits along the long-pitch helix of actin filaments. Once bound, tropomyosin polymerizes end-to-end and both stabilizes F-actin and regulates access of various actin binding proteins including myosin in muscle and non-muscle cells. Single tropomyosin molecules bind weakly to F-actin with millimolar Kd, whereas the end-to-end linked tropomyosin associates with about a one thousand-fold greater affinity. Despite years of study, the assembly mechanism of tropomyosin onto actin filaments remains unclear. In the current study, we used total internal reflection fluorescence (TIRF) microscopy to directly monitor the cooperative binding of fluorescently labeled tropomyosin molecules to phalloidin-stabilized actin filaments. We find that tropomyosin molecules assemble from multiple growth sites following random low affinity binding of single molecules to actin. As the length of the tropomyosin chain increases, the probability of detachment decreases, which leads to further chain growth. Tropomyosin chain extension is linearly dependent on tropomyosin concentration, occurring at approximately 100 monomers/(μM*s). The random tropomyosin binding to F-actin leads to discontinuous end-to-end association where gaps in the chain continuity smaller than the required seven sequential actin monomers are available. Direct observation of tropomyosin detachment revealed the number of gaps in actin-bound tropomyosin, the time course of gap annealing, and the eventual filament saturation process. PMID:26033920

  1. Actin in xenopus oocytes: II. intracellular distribution and polymerizability

    PubMed Central

    Merriam, RW; Clark, TG

    1978-01-01

    The largest oocytes of Xenopus Laevis were broken open in the absence of shearing forces which might transfer actin from particulate to supernatant fractions. Particulate and postmitochondrial supernatant fractions were prepared by centrifugation. SDS-electrophoretic fractionation on polyacrylamide gels and quantitative scanning techniques were used to separate actin and to assay its amount in cellular fractions. The actin has been identified in electrophoretograms by its molecular weight and its binding to DNase I. oocytes contain 1.4-1.7 {um}g of actin per cell, of which up to 88 percent is recovered in the postmitochondrial supernate under a variety of conditions. In the soluble fraction, it represents about 8.8 percent of the total protein. Its concentration in native cytoplasm was directly assayed at 4.1 mg/ml. There is no detectable actin that can be transferred from the particulate to the soluble phase by neutral detergents or ionic conditions that would depolymerize muscle actin. Centrifugation of the soluble oocyte fractions showed that 75-95 percent of the actin can not be sedimented under forces that would pellet filamentous actin. Addition of potassium and magnesium to the cytoplasm, to concentrations that would polymerize muscle actin, does not increase the amount of sedimentable actin. Roughly one-third of the soluble actin is recovered from Sephadex columns at about the position of monomer. About two- thirds is in complexes of 100,000 daltons or greater. PMID:565782

  2. Distributed actin turnover in the lamellipodium and FRAP kinetics.

    PubMed

    Smith, Matthew B; Kiuchi, Tai; Watanabe, Naoki; Vavylonis, Dimitrios

    2013-01-01

    Studies of actin dynamics at the leading edge of motile cells with single-molecule speckle (SiMS) microscopy have shown a broad distribution of EGFP-actin speckle lifetimes and indicated actin polymerization and depolymerization over an extended region. Other experiments using FRAP with the same EGFP-actin as a probe have suggested, by contrast, that polymerization occurs exclusively at the leading edge. We performed FRAP experiments on XTC cells to compare SiMS to FRAP on the same cell type. We used speckle statistics obtained by SiMS to model the steady-state distribution and kinetics of actin in the lamellipodium. We demonstrate that a model with a single diffuse actin species is in good agreement with FRAP experiments. A model including two species of diffuse actin provides an even better agreement. The second species consists of slowly diffusing oligomers that associate to the F-actin network throughout the lamellipodium or break up into monomers after a characteristic time. Our work motivates studies to test the presence and composition of slowly diffusing actin species that may contribute to local remodeling of the actin network and increase the amount of soluble actin.

  3. Bundling actin filaments from membranes: some novel players

    PubMed Central

    Thomas, Clément

    2012-01-01

    Progress in live-cell imaging of the cytoskeleton has significantly extended our knowledge about the organization and dynamics of actin filaments near the plasma membrane of plant cells. Noticeably, two populations of filamentous structures can be distinguished. On the one hand, fine actin filaments which exhibit an extremely dynamic behavior basically characterized by fast polymerization and prolific severing events, a process referred to as actin stochastic dynamics. On the other hand, thick actin bundles which are composed of several filaments and which are comparatively more stable although they constantly remodel as well. There is evidence that the actin cytoskeleton plays critical roles in trafficking and signaling at both the cell cortex and organelle periphery but the exact contribution of actin bundles remains unclear. A common view is that actin bundles provide the long-distance tracks used by myosin motors to deliver their cargo to growing regions and accordingly play a particularly important role in cell polarization. However, several studies support that actin bundles are more than simple passive highways and display multiple and dynamic roles in the regulation of many processes, such as cell elongation, polar auxin transport, stomatal and chloroplast movement, and defense against pathogens. The list of identified plant actin-bundling proteins is ever expanding, supporting that plant cells shape structurally and functionally different actin bundles. Here I review the most recently characterized actin-bundling proteins, with a particular focus on those potentially relevant to membrane trafficking and/or signaling. PMID:22936939

  4. A second Las17 monomeric actin-binding motif functions in Arp2/3-dependent actin polymerization during endocytosis.

    PubMed

    Feliciano, Daniel; Tolsma, Thomas O; Farrell, Kristen B; Aradi, Al; Di Pietro, Santiago M

    2015-04-01

    During clathrin-mediated endocytosis (CME), actin assembly provides force to drive vesicle internalization. Members of the Wiskott-Aldrich syndrome protein (WASP) family play a fundamental role stimulating actin assembly. WASP family proteins contain a WH2 motif that binds globular actin (G-actin) and a central-acidic motif that binds the Arp2/3 complex, thus promoting the formation of branched actin filaments. Yeast WASP (Las17) is the strongest of five factors promoting Arp2/3-dependent actin polymerization during CME. It was suggested that this strong activity may be caused by a putative second G-actin-binding motif in Las17. Here, we describe the in vitro and in vivo characterization of such Las17 G-actin-binding motif (LGM) and its dependence on a group of conserved arginine residues. Using the yeast two-hybrid system, GST-pulldown, fluorescence polarization and pyrene-actin polymerization assays, we show that LGM binds G-actin and is necessary for normal Arp2/3-mediated actin polymerization in vitro. Live-cell fluorescence microscopy experiments demonstrate that LGM is required for normal dynamics of actin polymerization during CME. Further, LGM is necessary for normal dynamics of endocytic machinery components that are recruited at early, intermediate and late stages of endocytosis, as well as for optimal endocytosis of native CME cargo. Both in vitro and in vivo experiments show that LGM has relatively lower potency compared to the previously known Las17 G-actin-binding motif, WH2. These results establish a second G-actin-binding motif in Las17 and advance our knowledge on the mechanism of actin assembly during CME.

  5. The Potential Roles of Actin in The Nucleus

    PubMed Central

    Falahzadeh, Khadijeh; Banaei-Esfahani, Amir; Shahhoseini, Maryam

    2015-01-01

    Over the past few decades, actin’s presence in the nucleus has been demonstrated. Actin is a key protein necessary for different nuclear processes. Although actin is well known for its functional role in dynamic behavior of the cytoskeleton, emerging studies are now highlighting new roles for actin. At the present time there is no doubt about the presence of actin in the nucleus. A number of studies have uncovered the functional involvement of actin in nuclear processes. Actin as one of the nuclear components has its own structured and functional rules, such as nuclear matrix association, chromatin remodeling, transcription by RNA polymerases I, II, III and mRNA processing. In this historical review, we attempt to provide an overview of our current understanding of the functions of actin in the nucleus. PMID:25870830

  6. Regulation of cellular actin architecture by S100A10.

    PubMed

    Jung, M Juliane; Murzik, Ulrike; Wehder, Liane; Hemmerich, Peter; Melle, Christian

    2010-04-15

    Actin structures are involved in several biological processes and the disruption of actin polymerisation induces impaired motility of eukaryotic cells. Different factors are involved in regulation and maintenance of the cytoskeletal actin architecture. Here we show that S100A10 participates in the particular organisation of actin filaments. Down-regulation of S100A10 by specific siRNA triggered a disorganisation of filamentous actin structures without a reduction of the total cellular actin concentration. In contrast, the formation of cytoskeleton structures containing tubulin was unhindered in S100A10 depleted cells. Interestingly, the cellular distribution of annexin A2, an interaction partner of S100A10, was unaffected in S100A10 depleted cells. Cells lacking S100A10 showed an impaired migration activity and were unable to close a scratched wound. Our data provide first insights of S100A10 function as a regulator of the filamentous actin network. PMID:20100475

  7. Partial Depletion of Gamma-Actin Suppresses Microtubule Dynamics

    PubMed Central

    Po'uha, Sela T; Honore, Stephane; Braguer, Diane; Kavallaris, Maria

    2013-01-01

    Actin and microtubule interactions are important for many cellular events, however these interactions are poorly described. Alterations in γ-actin are associated with diseases such as hearing loss and cancer. Functional investigations demonstrated that partial depletion of γ-actin affects cell polarity and induces resistance to microtubule-targeted agents. To determine whether γ-actin alterations directly affect microtubule dynamics, microtubule dynamic instability was analyzed in living cells following partial siRNA depletion of γ-actin. Partial depletion of γ-actin suppresses interphase microtubule dynamics by 17.5% due to a decrease in microtubule shortening rates and an increase in microtubule attenuation. γ-Actin partial depletion also increased distance-based microtubule catastrophe and rescue frequencies. In addition, knockdown of γ-actin delayed mitotic progression, partially blocking metaphase–anaphase transition and inhibiting cell proliferation. Interestingly, in the presence of paclitaxel, interphase microtubule dynamics were further suppressed by 24.4% in the γ-actin knockdown cells, which is comparable to 28.8% suppression observed in the control siRNA treated cells. Paclitaxel blocked metaphase–anaphase transition in both the γ-actin knockdown cells and the control siRNA cells. However, the extent of mitotic arrest was much higher in the control cells (28.4%), compared to the γ-actin depleted cells (8.5%). Therefore, suppression of microtubule dynamics by partial depletion of γ-actin is associated with marked delays in metaphase-anaphase transition and not mitotic arrest. This is the first demonstration that γ-actin can modulate microtubule dynamics by reducing the microtubule shortening rate, promoting paused/attenuated microtubules, and increasing transition frequencies suggesting a mechanistic link between γ-actin and microtubules. © 2013 Wiley Periodicals, Inc PMID:23335583

  8. Distinct Functional Interactions between Actin Isoforms and Nonsarcomeric Myosins

    PubMed Central

    Müller, Mirco; Diensthuber, Ralph P.; Chizhov, Igor; Claus, Peter; Heissler, Sarah M.; Preller, Matthias; Taft, Manuel H.; Manstein, Dietmar J.

    2013-01-01

    Despite their near sequence identity, actin isoforms cannot completely replace each other in vivo and show marked differences in their tissue-specific and subcellular localization. Little is known about isoform-specific differences in their interactions with myosin motors and other actin-binding proteins. Mammalian cytoplasmic β- and γ-actin interact with nonsarcomeric conventional myosins such as the members of the nonmuscle myosin-2 family and myosin-7A. These interactions support a wide range of cellular processes including cytokinesis, maintenance of cell polarity, cell adhesion, migration, and mechano-electrical transduction. To elucidate differences in the ability of isoactins to bind and stimulate the enzymatic activity of individual myosin isoforms, we characterized the interactions of human skeletal muscle α-actin, cytoplasmic β-actin, and cytoplasmic γ-actin with human myosin-7A and nonmuscle myosins-2A, -2B and -2C1. In the case of nonmuscle myosins-2A and -2B, the interaction with either cytoplasmic actin isoform results in 4-fold greater stimulation of myosin ATPase activity than was observed in the presence of α-skeletal muscle actin. Nonmuscle myosin-2C1 is most potently activated by β-actin and myosin-7A by γ-actin. Our results indicate that β- and γ-actin isoforms contribute to the modulation of nonmuscle myosin-2 and myosin-7A activity and thereby to the spatial and temporal regulation of cytoskeletal dynamics. FRET-based analyses show efficient copolymerization abilities for the actin isoforms in vitro. Experiments with hybrid actin filaments show that the extent of actomyosin coupling efficiency can be regulated by the isoform composition of actin filaments. PMID:23923011

  9. Reconstitution and Protein Composition Analysis of Endocytic Actin Patches

    PubMed Central

    Michelot, Alphée; Costanzo, Michael; Sarkeshik, Ali; Boone, Charles; Yates, John R.; Drubin, David G.

    2010-01-01

    Summary Background Clathrin-actin-mediated endocytosis in yeast involves the progressive assembly of at least 60 different proteins at cortical sites. More than half of these proteins are involved in the assembly of a branched network of actin filaments to provide the forces required for plasma membrane invagination. Results To gain insights into the regulation of endocytic actin patch dynamics, we developed an in vitro actin assembly assay using microbeads functionalized with the nucleation promoting factor (NPF) Las17 (yeast WASP). When incubated in a yeast extract, these beads assembled actin networks and a significant fraction became motile. Multi dimensional Protein Identification Technology (MudPIT) showed that the recruitment of actin binding proteins to these Las17-derived actin networks is selective. None of the proteins known to exclusively regulate the in vivo formation of actin cables or the actin contractile ring were identified. Intriguingly, our analysis also identified components of three other cortical structures, eisosomes, PIK patches and the TORC2 complex, establishing intriguing biochemical connections between four different yeast cortical complexes. Finally, we identified Aim3 as a regulator of actin dynamics at endocytic sites. Conclusions WASP is sufficient to trigger assembly of actin networks composed selectively of actin-patch proteins. These experiments establish that the protein composition of different F-actin structures is determined by the protein factor that initiates the network. The identification of binding partners revealed new biochemical connections between WASP derived networks and other cortical complexes and identified Aim3 as a novel regulator of the endocytic actin patch. PMID:21035341

  10. Caffeine relaxes smooth muscle through actin depolymerization.

    PubMed

    Tazzeo, Tracy; Bates, Genevieve; Roman, Horia Nicolae; Lauzon, Anne-Marie; Khasnis, Mukta D; Eto, Masumi; Janssen, Luke J

    2012-08-15

    Caffeine is sometimes used in cell physiological studies to release internally stored Ca(2+). We obtained evidence that caffeine may also act through a different mechanism that has not been previously described and sought to examine this in greater detail. We ruled out a role for phosphodiesterase (PDE) inhibition, since the effect was 1) not reversed by inhibiting PKA or adenylate cyclase; 2) not exacerbated by inhibiting PDE4; and 3) not mimicked by submillimolar caffeine nor theophylline, both of which are sufficient to inhibit PDE. Although caffeine is an agonist of bitter taste receptors, which in turn mediate bronchodilation, its relaxant effect was not mimicked by quinine. After permeabilizing the membrane using β-escin and depleting the internal Ca(2+) store using A23187, we found that 10 mM caffeine reversed tone evoked by direct application of Ca(2+), suggesting it functionally antagonizes the contractile apparatus. Using a variety of molecular techniques, we found that caffeine did not affect phosphorylation of myosin light chain (MLC) by MLC kinase, actin-filament motility catalyzed by MLC kinase, phosphorylation of CPI-17 by either protein kinase C or RhoA kinase, nor the activity of MLC-phosphatase. However, we did obtain evidence that caffeine decreased actin filament binding to phosphorylated myosin heads and increased the ratio of globular to filamentous actin in precontracted tissues. We conclude that, in addition to its other non-RyR targets, caffeine also interferes with actin function (decreased binding by myosin, possibly with depolymerization), an effect that should be borne in mind in studies using caffeine to probe excitation-contraction coupling in smooth muscle.

  11. Summertime actinic lichenoid eruption (lichen planus actinicus).

    PubMed

    Isaacson, D; Turner, M L; Elgart, M L

    1981-04-01

    A patient with an unusual lichenoid photosensitive eruption is presented. The features differentiating this entity from classic lichen planus, lupus erythematosus, and polymorphous light eruption are discussed. Phototesting confirmed the provocative influence of sunlight. We prefer the name "summertime actinic lichenoid eruption" to that of "lichen planus subtropicus" or "lichen planus actinicus," since this condition is not confined to subtropical countries, and, although light appears to initiate the eruption, its exact relationship to lichen planus is unclear.

  12. Viscoelastic properties of actin-coated membranes.

    PubMed

    Helfer, E; Harlepp, S; Bourdieu, L; Robert, J; MacKintosh, F C; Chatenay, D

    2001-02-01

    In living cells, cytoskeletal filaments interact with the plasma membrane to form structures that play a key role in cell shape and mechanical properties. To study the interaction between these basic components, we designed an in vitro self-assembled network of actin filaments attached to the outer surface of giant unilamellar vesicles. Optical tweezers and single-particle tracking experiments are used to study the rich dynamics of these actin-coated membranes (ACM). We show that microrheology studies can be carried out on such an individual microscopic object. The principle of the experiment consists in measuring the thermally excited position fluctuations of a probe bead attached biochemically to the membrane. We propose a model that relates the power spectrum of these thermal fluctuations to the viscoelastic properties of the membrane. The presence of the actin network modifies strongly the membrane dynamics with respect to a fluid, lipid bilayer one. It induces first a finite (omega=0) two-dimensional (2D) shear modulus G(0)(2D) approximately 0.5 to 5 microN/m in the membrane plane. Moreover, the frequency dependence at high frequency of the shear modulus [G(')(2D)(f ) approximately f(0.85+/-0.07)] and of the bending modulus (kappa(ACM)(f) approximately f(0.55+/-0.21)) demonstrate the viscoelastic behavior of the composite membrane. These results are consistent with a common exponent of 0.75 for both moduli as expected from our model and from prior measurements on actin solutions.

  13. Novel roles for actin in mitochondrial fission

    PubMed Central

    Hatch, Anna L.; Gurel, Pinar S.; Higgs, Henry N.

    2014-01-01

    ABSTRACT Mitochondrial dynamics, including fusion, fission and translocation, are crucial to cellular homeostasis, with roles in cellular polarity, stress response and apoptosis. Mitochondrial fission has received particular attention, owing to links with several neurodegenerative diseases. A central player in fission is the cytoplasmic dynamin-related GTPase Drp1, which oligomerizes at the fission site and hydrolyzes GTP to drive membrane ingression. Drp1 recruitment to the outer mitochondrial membrane (OMM) is a key regulatory event, which appears to require a pre-constriction step in which the endoplasmic reticulum (ER) and mitochondrion interact extensively, a process termed ERMD (ER-associated mitochondrial division). It is unclear how ER–mitochondrial contact generates the force required for pre-constriction or why pre-constriction leads to Drp1 recruitment. Recent results, however, show that ERMD might be an actin-based process in mammals that requires the ER-associated formin INF2 upstream of Drp1, and that myosin II and other actin-binding proteins might be involved. In this Commentary, we present a mechanistic model for mitochondrial fission in which actin and myosin contribute in two ways; firstly, by supplying the force for pre-constriction and secondly, by serving as a coincidence detector for Drp1 binding. In addition, we discuss the possibility that multiple fission mechanisms exist in mammals. PMID:25217628

  14. Virulent Burkholderia species mimic host actin polymerases to drive actin-based motility

    PubMed Central

    Benanti, Erin L.; Nguyen, Catherine M.; Welch, Matthew D.

    2015-01-01

    Summary Burkholderia pseudomallei and B. mallei are bacterial pathogens that cause melioidosis and glanders, while their close relative B. thailandensis is nonpathogenic. All use the trimeric autotransporter BimA to facilitate actin-based motility, host cell fusion and dissemination. Here, we show that BimA orthologs mimic different host actin-polymerizing proteins. B. thailandensis BimA activates the host Arp2/3 complex. In contrast, B. pseudomallei and B. mallei BimA mimic host Ena/VASP actin polymerases in their ability to nucleate, elongate and bundle filaments by associating with barbed ends, as well as in their use of WH2 motifs and oligomerization for activity. Mechanistic differences among BimA orthologs resulted in distinct actin filament organization and motility parameters, which affected the efficiency of cell fusion during infection. Our results identify bacterial Ena/VASP mimics and reveal that pathogens imitate the full spectrum of host actin-polymerizing pathways, suggesting that mimicry of different polymerization mechanisms influences key parameters of infection. PMID:25860613

  15. Virulent Burkholderia species mimic host actin polymerases to drive actin-based motility.

    PubMed

    Benanti, Erin L; Nguyen, Catherine M; Welch, Matthew D

    2015-04-01

    Burkholderia pseudomallei and B. mallei are bacterial pathogens that cause melioidosis and glanders, whereas their close relative B. thailandensis is non-pathogenic. All use the trimeric autotransporter BimA to facilitate actin-based motility, host cell fusion, and dissemination. Here, we show that BimA orthologs mimic different host actin-polymerizing proteins. B. thailandensis BimA activates the host Arp2/3 complex. In contrast, B. pseudomallei and B. mallei BimA mimic host Ena/VASP actin polymerases in their ability to nucleate, elongate, and bundle filaments by associating with barbed ends, as well as in their use of WH2 motifs and oligomerization for activity. Mechanistic differences among BimA orthologs resulted in distinct actin filament organization and motility parameters, which affected the efficiency of cell fusion during infection. Our results identify bacterial Ena/VASP mimics and reveal that pathogens imitate the full spectrum of host actin-polymerizing pathways, suggesting that mimicry of different polymerization mechanisms influences key parameters of infection.

  16. Lamellipodin promotes actin assembly by clustering Ena/VASP proteins and tethering them to actin filaments

    PubMed Central

    Hansen, Scott D; Mullins, R Dyche

    2015-01-01

    Enabled/Vasodilator (Ena/VASP) proteins promote actin filament assembly at multiple locations, including: leading edge membranes, focal adhesions, and the surface of intracellular pathogens. One important Ena/VASP regulator is the mig-10/Lamellipodin/RIAM family of adaptors that promote lamellipod formation in fibroblasts and drive neurite outgrowth and axon guidance in neurons. To better understand how MRL proteins promote actin network formation we studied the interactions between Lamellipodin (Lpd), actin, and VASP, both in vivo and in vitro. We find that Lpd binds directly to actin filaments and that this interaction regulates its subcellular localization and enhances its effect on VASP polymerase activity. We propose that Lpd delivers Ena/VASP proteins to growing barbed ends and increases their polymerase activity by tethering them to filaments. This interaction represents one more pathway by which growing actin filaments produce positive feedback to control localization and activity of proteins that regulate their assembly. DOI: http://dx.doi.org/10.7554/eLife.06585.001 PMID:26295568

  17. Dynacortin is a novel actin bundling protein that localizes to dynamic actin structures.

    PubMed

    Robinson, Douglas N; Ocon, Stephani S; Rock, Ronald S; Spudich, James A

    2002-03-15

    Dynacortin is a novel protein that was discovered in a genetic suppressor screen of a Dictyostelium discoideum cytokinesis-deficient mutant cell line devoid of the cleavage furrow actin bundling protein, cortexillin I. While dynacortin is highly enriched in the cortex, particularly in cell-surface protrusions, it is excluded from the cleavage furrow cortex during cytokinesis. Here, we describe the biochemical characterization of this new protein. Purified dynacortin is an 80-kDa dimer with a large 5.7-nm Stokes radius. Dynacortin cross-links actin filaments into parallel arrays with a mole ratio of one dimer to 1.3 actin monomers and a 3.1 microm K(d). Using total internal reflection fluorescence microscopy, GFP-dynacortin and the actin bundling protein coronin-GFP are seen to concentrate in highly dynamic cortical structures with assembly and disassembly half-lives of about 15 s. These results indicate that cells have evolved different actin-filament cross-linking proteins with complementary cellular distributions that collaborate to orchestrate complex cell shape changes.

  18. AkF ¯ chiral gauge theories

    NASA Astrophysics Data System (ADS)

    Shi, Yan-Liang; Shrock, Robert

    2015-11-01

    We study asymptotically free chiral gauge theories with an SU (N ) gauge group and chiral fermions transforming according to the antisymmetric rank-k tensor representation, Ak≡[k ]N , and the requisite number, nF ¯, of copies of fermions in the conjugate fundamental representation, F ¯ ≡[1] ¯ N , to render the theories anomaly-free. We denote these as AkF ¯ theories. We take N ≥2 k +1 so that nF ¯≥1 . The A2F ¯ theories form an infinite family with N ≥5 , but we show that the A3F ¯ and A4F ¯ theories are only asymptotically free for N in the respective ranges 7 ≤N ≤17 and 9 ≤N ≤11 , and that there are no asymptotically free AkF ¯ theories with k ≥5 . We investigate the types of ultraviolet to infrared evolution for these AkF ¯ theories and find that, depending on k and N , they may lead to a non-Abelian Coulomb phase, or may involve confinement with massless gauge-singlet composite fermions, bilinear fermion condensation with dynamical gauge and global symmetry breaking, or formation of multifermion condensates that preserve the gauge symmetry. We also show that there are no asymptotically free, anomaly-free SU (N ) SkF ¯ chiral gauge theories with k ≥3 , where Sk denotes the rank-k symmetric representation.

  19. Actin is required for IFT regulation in Chlamydomonas reinhardtii.

    PubMed

    Avasthi, Prachee; Onishi, Masayuki; Karpiak, Joel; Yamamoto, Ryosuke; Mackinder, Luke; Jonikas, Martin C; Sale, Winfield S; Shoichet, Brian; Pringle, John R; Marshall, Wallace F

    2014-09-01

    Assembly of cilia and flagella requires intraflagellar transport (IFT), a highly regulated kinesin-based transport system that moves cargo from the basal body to the tip of flagella [1]. The recruitment of IFT components to basal bodies is a function of flagellar length, with increased recruitment in rapidly growing short flagella [2]. The molecular pathways regulating IFT are largely a mystery. Because actin network disruption leads to changes in ciliary length and number, actin has been proposed to have a role in ciliary assembly. However, the mechanisms involved are unknown. In Chlamydomonas reinhardtii, conventional actin is found in both the cell body and the inner dynein arm complexes within flagella [3, 4]. Previous work showed that treating Chlamydomonas cells with the actin-depolymerizing compound cytochalasin D resulted in reversible flagellar shortening [5], but how actin is related to flagellar length or assembly remains unknown. Here we utilize small-molecule inhibitors and genetic mutants to analyze the role of actin dynamics in flagellar assembly in Chlamydomonas reinhardtii. We demonstrate that actin plays a role in IFT recruitment to basal bodies during flagellar elongation and that when actin is perturbed, the normal dependence of IFT recruitment on flagellar length is lost. We also find that actin is required for sufficient entry of IFT material into flagella during assembly. These same effects are recapitulated with a myosin inhibitor, suggesting that actin may act via myosin in a pathway by which flagellar assembly is regulated by flagellar length.

  20. Tropomyosin - master regulator of actin filament function in the cytoskeleton.

    PubMed

    Gunning, Peter W; Hardeman, Edna C; Lappalainen, Pekka; Mulvihill, Daniel P

    2015-08-15

    Tropomyosin (Tpm) isoforms are the master regulators of the functions of individual actin filaments in fungi and metazoans. Tpms are coiled-coil parallel dimers that form a head-to-tail polymer along the length of actin filaments. Yeast only has two Tpm isoforms, whereas mammals have over 40. Each cytoskeletal actin filament contains a homopolymer of Tpm homodimers, resulting in a filament of uniform Tpm composition along its length. Evidence for this 'master regulator' role is based on four core sets of observation. First, spatially and functionally distinct actin filaments contain different Tpm isoforms, and recent data suggest that members of the formin family of actin filament nucleators can specify which Tpm isoform is added to the growing actin filament. Second, Tpms regulate whole-organism physiology in terms of morphogenesis, cell proliferation, vesicle trafficking, biomechanics, glucose metabolism and organ size in an isoform-specific manner. Third, Tpms achieve these functional outputs by regulating the interaction of actin filaments with myosin motors and actin-binding proteins in an isoform-specific manner. Last, the assembly of complex structures, such as stress fibers and podosomes involves the collaboration of multiple types of actin filament specified by their Tpm composition. This allows the cell to specify actin filament function in time and space by simply specifying their Tpm isoform composition.

  1. The neuronal and actin commitment: Why do neurons need rings?

    PubMed

    Leite, Sérgio Carvalho; Sousa, Mónica Mendes

    2016-09-01

    The role of the actin cytoskeleton in neurons has been extensively studied in actin-enriched compartments such as the growth cone and dendritic spines. The recent discovery of actin rings in the axon shaft and in dendrites, together with the identification of axon actin trails, has advanced our understanding on actin organization and dynamics in neurons. However, specifically in the case of actin rings, the mechanisms regulating their nucleation and assembly, and the functions that they may exert in axons and dendrites remain largely unexplored. Here we discuss the possible structural, mechanistic and functional properties of the subcortical neuronal cytoskeleton putting the current knowledge in perspective with the information available on actin rings formed in other biological contexts, and with the organization of actin-spectrin lattices in other cell types. The detailed analysis of these novel neuronal actin ring structures, together with the elucidation of the function of actin-binding proteins in neuron biology, has a large potential to uncover new mechanisms of neuronal function under normal conditions that may have impact in our understanding of axon degeneration and regeneration. © 2016 Wiley Periodicals, Inc.

  2. Excitable actin dynamics in lamellipodial protrusion and retraction.

    PubMed

    Ryan, Gillian L; Petroccia, Heather M; Watanabe, Naoki; Vavylonis, Dimitrios

    2012-04-01

    Many animal cells initiate crawling by protruding lamellipodia, consisting of a dense network of actin filaments, at their leading edge. We imaged XTC cells that exhibit flat lamellipodia on poly-L-lysine-coated coverslips. Using active contours, we tracked the leading edge and measured the total amount of F-actin by summing the pixel intensities within a 5-μm band. We observed protrusion and retraction with period 130-200 s and local wavelike features. Positive (negative) velocities correlated with minimum (maximum) integrated actin concentration. Approximately constant retrograde flow indicated that protrusions and retractions were driven by fluctuations of the actin polymerization rate. We present a model of these actin dynamics as an excitable system in which a diffusive, autocatalytic activator causes actin polymerization; F-actin accumulation in turn inhibits further activator accumulation. Simulations of the model reproduced the pattern of actin polymerization seen in experiments. To explore the model's assumption of an autocatalytic activation mechanism, we imaged cells expressing markers for both F-actin and the p21 subunit of the Arp2/3 complex. We found that integrated Arp2/3-complex concentrations spike several seconds before spikes of F-actin concentration. This suggests that the Arp2/3 complex participates in an activation mechanism that includes additional diffuse components. Response of cells to stimulation by fetal calf serum could be reproduced by the model, further supporting the proposed dynamical picture.

  3. Effect of Tropomyosin on Formin-Bound Actin Filaments

    PubMed Central

    Ujfalusi, Zoltán; Vig, Andrea; Hild, Gábor; Nyitrai, Miklós

    2009-01-01

    Abstract Formins are conservative proteins with important roles in the regulation of the microfilament system in eukaryotic cells. Previous studies showed that the binding of formins to actin made the structure of actin filaments more flexible. Here, the effects of tropomyosin on formin-induced changes in actin filaments were investigated using fluorescence spectroscopic methods. The temperature dependence of the Förster-type resonance energy transfer showed that the formin-induced increase of flexibility of actin filaments was diminished by the binding of tropomyosin to actin. Fluorescence anisotropy decay measurements also revealed that the structure of flexible formin-bound actin filaments was stabilized by the binding of tropomyosin. The stabilizing effect reached its maximum when all binding sites on actin were occupied by tropomyosin. The effect of tropomyosin on actin filaments was independent of ionic strength, but became stronger as the magnesium concentration increased. Based on these observations, we propose that in cells there is a molecular mechanism in which tropomyosin binding to actin plays an important role in forming mechanically stable actin filaments, even in the case of formin-induced rapid filament assembly. PMID:18931257

  4. The neuronal and actin commitment: Why do neurons need rings?

    PubMed

    Leite, Sérgio Carvalho; Sousa, Mónica Mendes

    2016-09-01

    The role of the actin cytoskeleton in neurons has been extensively studied in actin-enriched compartments such as the growth cone and dendritic spines. The recent discovery of actin rings in the axon shaft and in dendrites, together with the identification of axon actin trails, has advanced our understanding on actin organization and dynamics in neurons. However, specifically in the case of actin rings, the mechanisms regulating their nucleation and assembly, and the functions that they may exert in axons and dendrites remain largely unexplored. Here we discuss the possible structural, mechanistic and functional properties of the subcortical neuronal cytoskeleton putting the current knowledge in perspective with the information available on actin rings formed in other biological contexts, and with the organization of actin-spectrin lattices in other cell types. The detailed analysis of these novel neuronal actin ring structures, together with the elucidation of the function of actin-binding proteins in neuron biology, has a large potential to uncover new mechanisms of neuronal function under normal conditions that may have impact in our understanding of axon degeneration and regeneration. © 2016 Wiley Periodicals, Inc. PMID:26784007

  5. The Yeast Actin Cytoskeleton: from Cellular Function to Biochemical Mechanism

    PubMed Central

    Moseley, James B.; Goode, Bruce L.

    2006-01-01

    All cells undergo rapid remodeling of their actin networks to regulate such critical processes as endocytosis, cytokinesis, cell polarity, and cell morphogenesis. These events are driven by the coordinated activities of a set of 20 to 30 highly conserved actin-associated proteins, in addition to many cell-specific actin-associated proteins and numerous upstream signaling molecules. The combined activities of these factors control with exquisite precision the spatial and temporal assembly of actin structures and ensure dynamic turnover of actin structures such that cells can rapidly alter their cytoskeletons in response to internal and external cues. One of the most exciting principles to emerge from the last decade of research on actin is that the assembly of architecturally diverse actin structures is governed by highly conserved machinery and mechanisms. With this realization, it has become apparent that pioneering efforts in budding yeast have contributed substantially to defining the universal mechanisms regulating actin dynamics in eukaryotes. In this review, we first describe the filamentous actin structures found in Saccharomyces cerevisiae (patches, cables, and rings) and their physiological functions, and then we discuss in detail the specific roles of actin-associated proteins and their biochemical mechanisms of action. PMID:16959963

  6. Tropomyosin - master regulator of actin filament function in the cytoskeleton.

    PubMed

    Gunning, Peter W; Hardeman, Edna C; Lappalainen, Pekka; Mulvihill, Daniel P

    2015-08-15

    Tropomyosin (Tpm) isoforms are the master regulators of the functions of individual actin filaments in fungi and metazoans. Tpms are coiled-coil parallel dimers that form a head-to-tail polymer along the length of actin filaments. Yeast only has two Tpm isoforms, whereas mammals have over 40. Each cytoskeletal actin filament contains a homopolymer of Tpm homodimers, resulting in a filament of uniform Tpm composition along its length. Evidence for this 'master regulator' role is based on four core sets of observation. First, spatially and functionally distinct actin filaments contain different Tpm isoforms, and recent data suggest that members of the formin family of actin filament nucleators can specify which Tpm isoform is added to the growing actin filament. Second, Tpms regulate whole-organism physiology in terms of morphogenesis, cell proliferation, vesicle trafficking, biomechanics, glucose metabolism and organ size in an isoform-specific manner. Third, Tpms achieve these functional outputs by regulating the interaction of actin filaments with myosin motors and actin-binding proteins in an isoform-specific manner. Last, the assembly of complex structures, such as stress fibers and podosomes involves the collaboration of multiple types of actin filament specified by their Tpm composition. This allows the cell to specify actin filament function in time and space by simply specifying their Tpm isoform composition. PMID:26240174

  7. Sequence and comparative genomic analysis of actin-related proteins.

    PubMed

    Muller, Jean; Oma, Yukako; Vallar, Laurent; Friederich, Evelyne; Poch, Olivier; Winsor, Barbara

    2005-12-01

    Actin-related proteins (ARPs) are key players in cytoskeleton activities and nuclear functions. Two complexes, ARP2/3 and ARP1/11, also known as dynactin, are implicated in actin dynamics and in microtubule-based trafficking, respectively. ARP4 to ARP9 are components of many chromatin-modulating complexes. Conventional actins and ARPs codefine a large family of homologous proteins, the actin superfamily, with a tertiary structure known as the actin fold. Because ARPs and actin share high sequence conservation, clear family definition requires distinct features to easily and systematically identify each subfamily. In this study we performed an in depth sequence and comparative genomic analysis of ARP subfamilies. A high-quality multiple alignment of approximately 700 complete protein sequences homologous to actin, including 148 ARP sequences, allowed us to extend the ARP classification to new organisms. Sequence alignments revealed conserved residues, motifs, and inserted sequence signatures to define each ARP subfamily. These discriminative characteristics allowed us to develop ARPAnno (http://bips.u-strasbg.fr/ARPAnno), a new web server dedicated to the annotation of ARP sequences. Analyses of sequence conservation among actins and ARPs highlight part of the actin fold and suggest interactions between ARPs and actin-binding proteins. Finally, analysis of ARP distribution across eukaryotic phyla emphasizes the central importance of nuclear ARPs, particularly the multifunctional ARP4.

  8. Dissociation of F-actin induced by hydrostatic pressure.

    PubMed

    Garcia, C R; Amaral Júnior, J A; Abrahamsohn, P; Verjovski-Almeida, S

    1992-11-01

    F-actin purified from rabbit skeletal muscle undergoes reversible dissociation when subjected to hydrostatic pressures up to 240 MPa. Dissociation and reversibility were detected by the following procedures: fluorescence spectral changes observed under pressure, when either intrinsic tryptophan or pyrenyl emission of N-(1-pyrenyl)iodoacetamide-labeled actin were monitored; electron microscopy of samples fixed under pressure; size-exclusion HPLC of pressurized actin. The effect of pressure upon F-actin that had been polymerized in the presence of either Mg2+, Ca2+ or K+ was studied. The standard volume changes for the association of actin subunits, calculated from pressure/dissociation curves were 74 +/- 14 ml/mol for Mg-F-actin, 79 +/- 12 ml/mol for Ca-F-actin and 328 +/- 63 ml/mol for K-F-actin, indicating that actin subunits are packed differently in the polymer depending on which cation is present. All pressure/dissociation data could be fitted by a model for dissociation of a dimer, which suggests that in the F-actin filament there is a predominant intersubunit interaction interface, most likely the head-to-tail intrastrand interaction between two subunits which repeats itself along the polymer. A tenfold change in total protein concentration from 20 micrograms to 200 micrograms/ml Mg-F-actin did not cause a change in the pressure required for half-maximal dissociation. This indicates a heterogeneity of free energy of association among actin monomers in the Mg-F-actin polymer, suggesting that, in addition to the predominant intersubunit interaction, the disordered interactions in the filament significantly contribute to the heterogeneity of microenvironments in the interface between the subunits. PMID:1425683

  9. Fibroblast-mediated contraction in actinically exposed and actinically protected aging skin

    SciTech Connect

    Marks, M.W.; Morykwas, M.J.; Wheatley, M.J. )

    1990-08-01

    The changes in skin morphology over time are a consequence of both chronologic aging and the accumulation of environmental exposure. Through observation, we know that actinic radiation intensifies the apparent aging of skin. We have investigated the effects of aging and actinic radiation on the ability of fibroblasts to contract collagen-fibroblast lattices. Preauricular and postauricular skin samples were obtained from eight patients aged 49 to 74 undergoing rhytidectomy. The samples were kept separate, and the fibroblasts were grown in culture. Lattices constructed with preauricular fibroblasts consistently contracted more than lattices containing postauricular fibroblasts. The difference in amount of contraction in 7 days between sites was greatest for the younger patients and decreased linearly as donor age increased (r = -0.96). This difference may be due to preauricular fibroblasts losing their ability to contract a lattice as aging skin is exposed to more actinic radiation.

  10. Photorhabdus luminescens toxins ADP-ribosylate actin and RhoA to force actin clustering.

    PubMed

    Lang, Alexander E; Schmidt, Gudula; Schlosser, Andreas; Hey, Timothy D; Larrinua, Ignacio M; Sheets, Joel J; Mannherz, Hans G; Aktories, Klaus

    2010-02-26

    The bacterium Photorhabdus luminescens is mutualistically associated with entomopathogenetic nematodes. These nematodes invade insect larvae and release the bacteria from their intestine, which kills the insects through the action of toxin complexes. We elucidated the mode of action of two of these insecticidal toxins from P. luminescens. We identified the biologically active components TccC3 and TccC5 as adenosine diphosphate (ADP)-ribosyltransferases, which modify unusual amino acids. TccC3 ADP-ribosylated threonine-148 of actin, resulting in actin polymerization. TccC5 ADP-ribosylated Rho guanosine triphosphatase proteins at glutamine-61 and glutamine-63, inducing their activation. The concerted action of both toxins inhibited phagocytosis of target insect cells and induced extensive intracellular polymerization and clustering of actin. Several human pathogenic bacteria produce related toxins. PMID:20185726

  11. 40 CFR Appendixes A-K to Part 51 - [Reserved

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 2 2011-07-01 2011-07-01 false A Appendixes A-K to Part 51 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS REQUIREMENTS FOR PREPARATION, ADOPTION, AND SUBMITTAL OF IMPLEMENTATION PLANS Appendixes A-K to Part 51...

  12. 40 CFR Appendixes A-K to Part 51 - [Reserved

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 2 2014-07-01 2014-07-01 false A Appendixes A-K to Part 51 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS REQUIREMENTS FOR PREPARATION, ADOPTION, AND SUBMITTAL OF IMPLEMENTATION PLANS Appendixes A-K to Part 51...

  13. 40 CFR Appendixes A-K to Part 51 - [Reserved

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 2 2010-07-01 2010-07-01 false A Appendixes A-K to Part 51 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS REQUIREMENTS FOR PREPARATION, ADOPTION, AND SUBMITTAL OF IMPLEMENTATION PLANS Appendixes A-K to Part 51...

  14. 40 CFR Appendixes A-K to Part 51 - [Reserved

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 2 2013-07-01 2013-07-01 false A Appendixes A-K to Part 51 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS REQUIREMENTS FOR PREPARATION, ADOPTION, AND SUBMITTAL OF IMPLEMENTATION PLANS Appendixes A-K to Part 51...

  15. 40 CFR Appendixes A-K to Part 51 - [Reserved

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 2 2012-07-01 2012-07-01 false A Appendixes A-K to Part 51 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS REQUIREMENTS FOR PREPARATION, ADOPTION, AND SUBMITTAL OF IMPLEMENTATION PLANS Appendixes A-K to Part 51...

  16. 46 CFR 7.180 - Kotzebue Sound, AK.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 1 2010-10-01 2010-10-01 false Kotzebue Sound, AK. 7.180 Section 7.180 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY PROCEDURES APPLICABLE TO THE PUBLIC BOUNDARY LINES Alaska § 7.180 Kotzebue Sound, AK. A line drawn from Cape Espenberg Light to latitude 66°52′ N. longitude 163°28′ W.;...

  17. 46 CFR 7.180 - Kotzebue Sound, AK.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 1 2013-10-01 2013-10-01 false Kotzebue Sound, AK. 7.180 Section 7.180 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY PROCEDURES APPLICABLE TO THE PUBLIC BOUNDARY LINES Alaska § 7.180 Kotzebue Sound, AK. A line drawn from Cape Espenberg Light to latitude 66°52′ N. longitude 163°28′ W.;...

  18. 46 CFR 7.180 - Kotzebue Sound, AK.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 1 2012-10-01 2012-10-01 false Kotzebue Sound, AK. 7.180 Section 7.180 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY PROCEDURES APPLICABLE TO THE PUBLIC BOUNDARY LINES Alaska § 7.180 Kotzebue Sound, AK. A line drawn from Cape Espenberg Light to latitude 66°52′ N. longitude 163°28′ W.;...

  19. 46 CFR 7.180 - Kotzebue Sound, AK.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 1 2014-10-01 2014-10-01 false Kotzebue Sound, AK. 7.180 Section 7.180 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY PROCEDURES APPLICABLE TO THE PUBLIC BOUNDARY LINES Alaska § 7.180 Kotzebue Sound, AK. A line drawn from Cape Espenberg Light to latitude 66°52′ N. longitude 163°28′ W.;...

  20. 46 CFR 7.180 - Kotzebue Sound, AK.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 1 2011-10-01 2011-10-01 false Kotzebue Sound, AK. 7.180 Section 7.180 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY PROCEDURES APPLICABLE TO THE PUBLIC BOUNDARY LINES Alaska § 7.180 Kotzebue Sound, AK. A line drawn from Cape Espenberg Light to latitude 66°52′ N. longitude 163°28′ W.;...

  1. Actin-Dynamics in Plant Cells: The Function of Actin Perturbing Substances Jasplakinolide, Chondramides, Phalloidin, Cytochalasins, and Latrunculins

    PubMed Central

    Holzinger, Andreas; Blaas, Kathrin

    2016-01-01

    This chapter will give an overview of the most common F-actin perturbing substances, that are used to study actin dynamics in living plant cells in studies on morphogenesis, motility, organelle movement or when apoptosis has to be induced. These substances can be divided into two major subclasses – F-actin stabilizing and polymerizing substances like jasplakinolide, chondramides and F-actin severing compounds like chytochalasins and latrunculins. Jasplakinolide was originally isolated form a marine sponge, and can now be synthesized and has become commercially available, which is responsible for its wide distribution as membrane permeable F-actin stabilizing and polymerizing agent, which may even have anti-cancer activities. Cytochalasins, derived from fungi show an F-actin severing function and many derivatives are commercially available (A, B, C, D, E, H, J), also making it a widely used compound for F-actin disruption. The same can be stated for latrunculins (A, B), derived from red sea sponges, however the mode of action is different by binding to G-actin and inhibiting incorporation into the filament. In the case of swinholide a stable complex with actin dimers is formed resulting also in severing of F-actin. For influencing F-actin dynamics in plant cells only membrane permeable drugs are useful in a broad range. We however introduce also the phallotoxins and synthetic derivatives, as they are widely used to visualize F-actin in fixed cells. A particular uptake mechanism has been shown for hepatocytes, but has also been described in siphonal giant algae. In the present chapter the focus is set on F-actin dynamics in plant cells where alterations in cytoplasmic streaming can be particularly well studied; however methods by fluorescence applications including phalloidin- and antibody staining as well as immunofluorescence-localization of the inhibitor drugs are given. PMID:26498789

  2. Identification of sucrose synthase as an actin-binding protein

    NASA Technical Reports Server (NTRS)

    Winter, H.; Huber, J. L.; Huber, S. C.; Davies, E. (Principal Investigator)

    1998-01-01

    Several lines of evidence indicate that sucrose synthase (SuSy) binds both G- and F-actin: (i) presence of SuSy in the Triton X-100-insoluble fraction of microsomal membranes (i.e. crude cytoskeleton fraction); (ii) co-immunoprecipitation of actin with anti-SuSy monoclonal antibodies; (iii) association of SuSy with in situ phalloidin-stabilized F-actin filaments; and (iv) direct binding to F-actin, polymerized in vitro. Aldolase, well known to interact with F-actin, interfered with binding of SuSy, suggesting that a common or overlapping binding site may be involved. We postulate that some of the soluble SuSy in the cytosol may be associated with the actin cytoskeleton in vivo.

  3. Spatial control of actin polymerization during neutrophil chemotaxis

    PubMed Central

    Weiner, Orion D.; Servant, Guy; Welch, Matthew D.; Mitchison, Timothy J.; Sedat, John W.; Bourne, Henry R.

    2010-01-01

    Neutrophils respond to chemotactic stimuli by increasing the nucleation and polymerization of actin filaments, but the location and regulation of these processes are not well understood. Here, using a permeabilized-cell assay, we show that chemotactic stimuli cause neutrophils to organize many discrete sites of actin polymerization, the distribution of which is biased by external chemotactic gradients. Furthermore, the Arp2/3 complex, which can nucleate actin polymerization, dynamically redistributes to the region of living neutrophils that receives maximal chemotactic stimulation, and the least-extractable pool of the Arp2/3 complex co-localizes with sites of actin polymerization. Our observations indicate that chemoattractant-stimulated neutrophils may establish discrete foci of actin polymerization that are similar to those generated at the posterior surface of the intracellular bacterium Listeria monocytogenes. We propose that asymmetrical establishment and/or maintenance of sites of actin polymerization produces directional migration of neutrophils in response to chemotactic gradients. PMID:10559877

  4. Reliable amplification of actin genes facilitates deep-level phylogeny.

    PubMed

    Voigt, K; Wöstemeyer, J

    2000-09-01

    The gene for actin as a highly conserved and functionally essential genetic element is developing into a major tool for phylogenetic analysis within a broad organismic range. We therefore propose a set of universally applicable primers that allow reliable amplification of actin genes. For primer construction the amino acid sequences of 57 actin genes comprising fungi, animals, plants and protists were analysed, aligned and used for the definition of six well-conserved regions which are suitable as priming sites in PCR amplification experiments. Ten primers were designed for specific in vitro amplification of actin gene fragments from a wide range of microorganisms. The corresponding gene fragments provide a strong basis to isolate nearly complete actin genes for further molecular characterization and for establishing phylogenies based on actin gene trees.

  5. Actin Depolymerization Drives Actomyosin Ring Contraction during Budding Yeast Cytokinesis

    PubMed Central

    Pinto, Inês Mendes; Rubinstein, Boris; Kucharavy, Andrei; Unruh, Jay R.; Li, Rong

    2012-01-01

    SUMMARY Actin filaments and myosin-II are evolutionarily conserved force generating components of the contractile ring during cytokinesis. Here we show that in budding yeast actin filament depolymerization plays a major role in actomyosin ring constriction. Cofilin mutation or chemically stabilizing actin filaments attenuates actomyosin ring constriction. Deletion of myosin-II motor domain or the myosin regulatory light chain reduced the contraction rate and also the rate of actin depolymerization in the ring. We constructed a quantitative microscopic model of actomyosin ring constriction via filament sliding driven by both actin depolymerization and myosin-II motor activity. Model simulations based on experimental measurements supports the notion that actin depolymerization is the predominant mechanism for ring constriction. The model predicts invariability of total contraction time irrespective of the initial ring size as originally reported for C elegans embryonic cells. This prediction was validated in yeast cells of different sizes due to having different ploidies. PMID:22698284

  6. Measuring F-actin properties in dendritic spines

    PubMed Central

    Koskinen, Mikko; Hotulainen, Pirta

    2014-01-01

    During the last decade, numerous studies have demonstrated that the actin cytoskeleton plays a pivotal role in the control of dendritic spine shape. Synaptic stimulation rapidly changes the actin dynamics and many actin regulators have been shown to play roles in neuron functionality. Accordingly, defects in the regulation of the actin cytoskeleton in neurons have been implicated in memory disorders. Due to the small size of spines, it is difficult to detect changes in the actin structures in dendritic spines by conventional light microscopy imaging. Instead, to know how tightly actin filaments are bundled together, and how fast the filaments turnover, we need to use advanced microscopy techniques, such as fluorescence recovery after photobleaching (FRAP), photoactivatable green fluorescent protein (PAGFP) fluorescence decay and fluorescence anisotropy. Fluorescence anisotropy, which measures the Förster resonance energy transfer (FRET) between two GFP fluorophores, has been proposed as a method to measure the level of actin polymerization. Here, we propose a novel idea that fluorescence anisotropy could be more suitable to study the level of actin filament bundling instead of actin polymerization. We validate the method in U2OS cell line where the actin structures can be clearly distinguished and apply to analyze how actin filament organization in dendritic spines changes during neuronal maturation. In addition to fluorescence anisotropy validation, we take a critical look at the properties and limitations of FRAP and PAGFP fluorescence decay methods and offer our proposals for the analysis methods for these approaches. These three methods complement each other, each providing additional information about actin dynamics and organization in dendritic spines. PMID:25140131

  7. The architecture of actin filaments and the ultrastructural location of actin-binding protein in the periphery of lung macrophages.

    PubMed

    Hartwig, J H; Shevlin, P

    1986-09-01

    A highly branched filament network is the principal structure in the periphery of detergent-extracted cytoskeletons of macrophages that have been spread on a surface and either freeze or critical point dried, and then rotary shadowed with platinum-carbon. This array of filaments completely fills lamellae extended from the cell and bifurcates to form 0.2-0.5 micron thick layers on the top and bottom of the cell body. Reaction of the macrophage cytoskeletons with anti-actin IgG and with anti-IgG bound to colloidal gold produces dense staining of these filaments, and incubation with myosin subfragment 1 uniformly decorates these filaments, identifying them as actin. 45% of the total cellular actin and approximately 70% of actin-binding protein remains in the detergent-insoluble cell residue. The soluble actin is not filamentous as determined by sedimentation analysis, the DNAase I inhibition assay, and electron microscopy, indicating that the cytoskeleton is not fragmented by detergent extraction. The spacing between the ramifications of the actin network is 94 +/- 47 nm and 118 +/- 72 nm in cytoskeletons prepared for electron microscopy by freeze drying and critical point drying, respectively. Free filament ends are rare, except for a few which project upward from the body of the network or which extend down to the substrate. Filaments of the network intersect predominantly at right angles to form either T-shaped and X-shaped overlaps having striking perpendicularity or else Y-shaped intersections composed of filaments intersecting at 120-130 degrees angles. The actin filament concentration in the lamellae is high, with an average value of 12.5 mg/ml. The concentration was much more uniform in freeze-dried preparations than in critical point-dried specimens, indicating that there is less collapse associated with the freezing technique. The orthogonal actin network of the macrophage cortical cytoplasm resembles actin gels made with actin-binding protein. Reaction of

  8. A Robust Actin Filaments Image Analysis Framework

    PubMed Central

    Alioscha-Perez, Mitchel; Benadiba, Carine; Goossens, Katty; Kasas, Sandor; Dietler, Giovanni; Willaert, Ronnie; Sahli, Hichem

    2016-01-01

    The cytoskeleton is a highly dynamical protein network that plays a central role in numerous cellular physiological processes, and is traditionally divided into three components according to its chemical composition, i.e. actin, tubulin and intermediate filament cytoskeletons. Understanding the cytoskeleton dynamics is of prime importance to unveil mechanisms involved in cell adaptation to any stress type. Fluorescence imaging of cytoskeleton structures allows analyzing the impact of mechanical stimulation in the cytoskeleton, but it also imposes additional challenges in the image processing stage, such as the presence of imaging-related artifacts and heavy blurring introduced by (high-throughput) automated scans. However, although there exists a considerable number of image-based analytical tools to address the image processing and analysis, most of them are unfit to cope with the aforementioned challenges. Filamentous structures in images can be considered as a piecewise composition of quasi-straight segments (at least in some finer or coarser scale). Based on this observation, we propose a three-steps actin filaments extraction methodology: (i) first the input image is decomposed into a ‘cartoon’ part corresponding to the filament structures in the image, and a noise/texture part, (ii) on the ‘cartoon’ image, we apply a multi-scale line detector coupled with a (iii) quasi-straight filaments merging algorithm for fiber extraction. The proposed robust actin filaments image analysis framework allows extracting individual filaments in the presence of noise, artifacts and heavy blurring. Moreover, it provides numerous parameters such as filaments orientation, position and length, useful for further analysis. Cell image decomposition is relatively under-exploited in biological images processing, and our study shows the benefits it provides when addressing such tasks. Experimental validation was conducted using publicly available datasets, and in osteoblasts

  9. A Robust Actin Filaments Image Analysis Framework.

    PubMed

    Alioscha-Perez, Mitchel; Benadiba, Carine; Goossens, Katty; Kasas, Sandor; Dietler, Giovanni; Willaert, Ronnie; Sahli, Hichem

    2016-08-01

    The cytoskeleton is a highly dynamical protein network that plays a central role in numerous cellular physiological processes, and is traditionally divided into three components according to its chemical composition, i.e. actin, tubulin and intermediate filament cytoskeletons. Understanding the cytoskeleton dynamics is of prime importance to unveil mechanisms involved in cell adaptation to any stress type. Fluorescence imaging of cytoskeleton structures allows analyzing the impact of mechanical stimulation in the cytoskeleton, but it also imposes additional challenges in the image processing stage, such as the presence of imaging-related artifacts and heavy blurring introduced by (high-throughput) automated scans. However, although there exists a considerable number of image-based analytical tools to address the image processing and analysis, most of them are unfit to cope with the aforementioned challenges. Filamentous structures in images can be considered as a piecewise composition of quasi-straight segments (at least in some finer or coarser scale). Based on this observation, we propose a three-steps actin filaments extraction methodology: (i) first the input image is decomposed into a 'cartoon' part corresponding to the filament structures in the image, and a noise/texture part, (ii) on the 'cartoon' image, we apply a multi-scale line detector coupled with a (iii) quasi-straight filaments merging algorithm for fiber extraction. The proposed robust actin filaments image analysis framework allows extracting individual filaments in the presence of noise, artifacts and heavy blurring. Moreover, it provides numerous parameters such as filaments orientation, position and length, useful for further analysis. Cell image decomposition is relatively under-exploited in biological images processing, and our study shows the benefits it provides when addressing such tasks. Experimental validation was conducted using publicly available datasets, and in osteoblasts grown in

  10. Correlative nanoscale imaging of actin filaments and their complexes

    PubMed Central

    Zhu, Huanqi; Grintsevich, Elena E.; Reisler, Emil

    2014-01-01

    Actin remodeling is an area of interest in biology in which correlative microscopy can bring a new way to analyze protein complexes at the nanoscale. Advances in EM, X-ray diffraction, fluorescence, and single molecule techniques have provided a wealth of information about the modulation of the F-actin structure and its regulation by actin binding proteins (ABPs). Yet, there are technological limitations of these approaches to achieving quantitative molecular level information on the structural and biophysical changes resulting from ABPs interaction with F-actin. Fundamental questions about the actin structure and dynamics and how these determine the function of ABPs remain unanswered. Specifically, how local and long-range structural and conformational changes result in ABPs induced remodeling of F-actin needs to be addressed at the single filament level. Advanced, sensitive and accurate experimental tools for detailed understanding of ABP–actin interactions are much needed. This article discusses the current understanding of nanoscale structural and mechanical modulation of F-actin by ABPs at the single filament level using several correlative microscopic techniques, focusing mainly on results obtained by Atomic Force Microscopy (AFM) analysis of ABP–actin complexes. PMID:23727693

  11. Human cytoplasmic actin proteins are encoded by a multigene family

    SciTech Connect

    Engel, J.; Gunning, P.; Kedes, L.

    1982-06-01

    The authors characterized nine human actin genes that they isolated from a library of cloned human DNA. Measurements of the thermal stability of hybrids formed between each cloned actin gene and ..cap alpha..-, ..beta..-, and ..gamma..-actin mRNA demonstrated that only one of the clones is most homologous to sarcomeric actin mRNA, whereas the remaining eight clones are most homologous to cytoplasmic actin mRNA. By the following criteria they show that these nine clones represent nine different actin gene loci rather than different alleles or different parts of a single gene: (i) the restriction enzyme maps of the coding regions are dissimilar; (ii) each clone contains sufficient coding region to encode all or most of an entire actin gene; and (iii) each clone contains sequences homologous to both the 5' and 3' ends of the coding region of a cloned chicken ..beta..-actin cDNA. They conclude, therefore, that the human cytoplasmic actin proteins are encoded by a multigene family.

  12. Solubilization of native actin monomers from human erythrocyte membranes.

    PubMed

    Tilley, L; Dwyer, M; Ralston, G B

    1986-01-01

    Up to 50% of the actin in erythrocyte membranes can be solubilized at low ionic strength in a form capable of inhibiting DNAse I, in the presence of 0.4 mM ATP and 0.05 mM calcium. In the absence of calcium and ATP, actin is released but is apparently rapidly denatured. Solubilization of G-actin increases with temperature up to 37 degrees C. At higher temperatures, actin is released rapidly but quickly loses its ability to inhibit DNAse I. PMID:3789986

  13. An unconventional form of actin in protozoan hemoflagellate, Leishmania.

    PubMed

    Kapoor, Prabodh; Sahasrabuddhe, Amogh A; Kumar, Ashutosh; Mitra, Kalyan; Siddiqi, Mohammad Imran; Gupta, Chhitar M

    2008-08-15

    Leishmania actin was cloned, overexpressed in baculovirus-insect cell system, and purified to homogeneity. The purified protein polymerized optimally in the presence of Mg2+ and ATP, but differed from conventional actins in its following properties: (i) it did not polymerize in the presence of Mg2+ alone, (ii) it polymerized in a restricted range of pH 7.0-8.5, (iii) its critical concentration for polymerization was found to be 3-4-fold lower than of muscle actin, (iv) it predominantly formed bundles rather than single filaments at pH 8.0, (v) it displayed considerably higher ATPase activity during polymerization, (vi) it did not inhibit DNase-I activity, and (vii) it did not bind the F-actin-binding toxin phalloidin or the actin polymerization disrupting agent Latrunculin B. Computational and molecular modeling studies revealed that the observed unconventional behavior of Leishmania actin is related to the diverged amino acid stretches in its sequence, which may lead to changes in the overall charge distribution on its solvent-exposed surface, ATP binding cleft, Mg2+ binding sites, and the hydrophobic loop that is involved in monomer-monomer interactions. Phylogenetically, it is related to ciliate actins, but to the best of our knowledge, no other actin with such unconventional properties has been reported to date. It is therefore suggested that actin in Leishmania may serve as a novel target for design of new antileishmanial drugs. PMID:18539603

  14. Actin in dendritic spines: connecting dynamics to function

    PubMed Central

    2010-01-01

    Dendritic spines are small actin-rich protrusions from neuronal dendrites that form the postsynaptic part of most excitatory synapses and are major sites of information processing and storage in the brain. Changes in the shape and size of dendritic spines are correlated with the strength of excitatory synaptic connections and heavily depend on remodeling of its underlying actin cytoskeleton. Emerging evidence suggests that most signaling pathways linking synaptic activity to spine morphology influence local actin dynamics. Therefore, specific mechanisms of actin regulation are integral to the formation, maturation, and plasticity of dendritic spines and to learning and memory. PMID:20457765

  15. Helical buckling of actin inside filopodia generates traction

    PubMed Central

    Leijnse, Natascha; Oddershede, Lene B.; Bendix, Poul M.

    2015-01-01

    Cells can interact with their surroundings via filopodia, which are membrane protrusions that extend beyond the cell body. Filopodia are essential during dynamic cellular processes like motility, invasion, and cell–cell communication. Filopodia contain cross-linked actin filaments, attached to the surrounding cell membrane via protein linkers such as integrins. These actin filaments are thought to play a pivotal role in force transduction, bending, and rotation. We investigated whether, and how, actin within filopodia is responsible for filopodia dynamics by conducting simultaneous force spectroscopy and confocal imaging of F-actin in membrane protrusions. The actin shaft was observed to periodically undergo helical coiling and rotational motion, which occurred simultaneously with retrograde movement of actin inside the filopodium. The cells were found to retract beads attached to the filopodial tip, and retraction was found to correlate with rotation and coiling of the actin shaft. These results suggest a previously unidentified mechanism by which a cell can use rotation of the filopodial actin shaft to induce coiling and hence axial shortening of the filopodial actin bundle. PMID:25535347

  16. Force generation by endocytic actin patches in budding yeast.

    PubMed

    Carlsson, Anders E; Bayly, Philip V

    2014-04-15

    Membrane deformation during endocytosis in yeast is driven by local, templated assembly of a sequence of proteins including polymerized actin and curvature-generating coat proteins such as clathrin. Actin polymerization is required for successful endocytosis, but it is not known by what mechanisms actin polymerization generates the required pulling forces. To address this issue, we develop a simulation method in which the actin network at the protein patch is modeled as an active gel. The deformation of the gel is treated using a finite-element approach. We explore the effects and interplay of three different types of force driving invagination: 1), forces perpendicular to the membrane, generated by differences between actin polymerization rates at the edge of the patch and those at the center; 2), the inherent curvature of the coat-protein layer; and 3), forces parallel to the membrane that buckle the coat protein layer, generated by an actomyosin contractile ring. We find that with optimistic estimates for the stall stress of actin gel growth and the shear modulus of the actin gel, actin polymerization can generate almost enough force to overcome the turgor pressure. In combination with the other mechanisms, actin polymerization can the force over the critical value.

  17. Force Generation by Endocytic Actin Patches in Budding Yeast

    PubMed Central

    Carlsson, Anders E.; Bayly, Philip V.

    2014-01-01

    Membrane deformation during endocytosis in yeast is driven by local, templated assembly of a sequence of proteins including polymerized actin and curvature-generating coat proteins such as clathrin. Actin polymerization is required for successful endocytosis, but it is not known by what mechanisms actin polymerization generates the required pulling forces. To address this issue, we develop a simulation method in which the actin network at the protein patch is modeled as an active gel. The deformation of the gel is treated using a finite-element approach. We explore the effects and interplay of three different types of force driving invagination: 1), forces perpendicular to the membrane, generated by differences between actin polymerization rates at the edge of the patch and those at the center; 2), the inherent curvature of the coat-protein layer; and 3), forces parallel to the membrane that buckle the coat protein layer, generated by an actomyosin contractile ring. We find that with optimistic estimates for the stall stress of actin gel growth and the shear modulus of the actin gel, actin polymerization can generate almost enough force to overcome the turgor pressure. In combination with the other mechanisms, actin polymerization can the force over the critical value. PMID:24739159

  18. Correlative nanoscale imaging of actin filaments and their complexes.

    PubMed

    Sharma, Shivani; Zhu, Huanqi; Grintsevich, Elena E; Reisler, Emil; Gimzewski, James K

    2013-07-01

    Actin remodeling is an area of interest in biology in which correlative microscopy can bring a new way to analyze protein complexes at the nanoscale. Advances in EM, X-ray diffraction, fluorescence, and single molecule techniques have provided a wealth of information about the modulation of the F-actin structure and its regulation by actin binding proteins (ABPs). Yet, there are technological limitations of these approaches to achieving quantitative molecular level information on the structural and biophysical changes resulting from ABPs interaction with F-actin. Fundamental questions about the actin structure and dynamics and how these determine the function of ABPs remain unanswered. Specifically, how local and long-range structural and conformational changes result in ABPs induced remodeling of F-actin needs to be addressed at the single filament level. Advanced, sensitive and accurate experimental tools for detailed understanding of ABP-actin interactions are much needed. This article discusses the current understanding of nanoscale structural and mechanical modulation of F-actin by ABPs at the single filament level using several correlative microscopic techniques, focusing mainly on results obtained by Atomic Force Microscopy (AFM) analysis of ABP-actin complexes.

  19. Arp2/3 complex and actin dynamics are required for actin-based mitochondrial motility in yeast.

    PubMed

    Boldogh, I R; Yang, H C; Nowakowski, W D; Karmon, S L; Hays, L G; Yates, J R; Pon, L A

    2001-03-13

    The Arp2/3 complex is implicated in actin polymerization-driven movement of Listeria monocytogenes. Here, we find that Arp2p and Arc15p, two subunits of this complex, show tight, actin-independent association with isolated yeast mitochondria. Arp2p colocalizes with mitochondria. Consistent with this result, we detect Arp2p-dependent formation of actin clouds around mitochondria in intact yeast. Cells bearing mutations in ARP2 or ARC15 genes show decreased velocities of mitochondrial movement, loss of all directed movement and defects in mitochondrial morphology. Finally, we observe a decrease in the velocity and extent of mitochondrial movement in yeast in which actin dynamics are reduced but actin cytoskeletal structure is intact. These results support the idea that the movement of mitochondria in yeast is actin polymerization driven and that this movement requires Arp2/3 complex.

  20. Visualization of Actin Cytoskeletal Dynamics in Fixed and Live Drosophila Egg Chambers.

    PubMed

    Groen, Christopher M; Tootle, Tina L

    2015-01-01

    Visualization of actin cytoskeletal dynamics is critical for understanding the spatial and temporal regulation of actin remodeling. Drosophila oogenesis provides an excellent model system for visualizing the actin cytoskeleton. Here, we present methods for imaging the actin cytoskeleton in Drosophila egg chambers in both fixed samples by phalloidin staining and in live egg chambers using transgenic actin labeling tools.

  1. A Mechanism for Actin Filament Severing by Malaria Parasite Actin Depolymerizing Factor 1 via a Low Affinity Binding Interface*

    PubMed Central

    Wong, Wilson; Webb, Andrew I.; Olshina, Maya A.; Infusini, Giuseppe; Tan, Yan Hong; Hanssen, Eric; Catimel, Bruno; Suarez, Cristian; Condron, Melanie; Angrisano, Fiona; NebI, Thomas; Kovar, David R.; Baum, Jake

    2014-01-01

    Actin depolymerizing factor (ADF)/cofilins are essential regulators of actin turnover in eukaryotic cells. These multifunctional proteins facilitate both stabilization and severing of filamentous (F)-actin in a concentration-dependent manner. At high concentrations ADF/cofilins bind stably to F-actin longitudinally between two adjacent actin protomers forming what is called a decorative interaction. Low densities of ADF/cofilins, in contrast, result in the optimal severing of the filament. To date, how these two contrasting modalities are achieved by the same protein remains uncertain. Here, we define the proximate amino acids between the actin filament and the malaria parasite ADF/cofilin, PfADF1 from Plasmodium falciparum. PfADF1 is unique among ADF/cofilins in being able to sever F-actin but do so without stable filament binding. Using chemical cross-linking and mass spectrometry (XL-MS) combined with structure reconstruction we describe a previously overlooked binding interface on the actin filament targeted by PfADF1. This site is distinct from the known binding site that defines decoration. Furthermore, total internal reflection fluorescence (TIRF) microscopy imaging of single actin filaments confirms that this novel low affinity site is required for F-actin severing. Exploring beyond malaria parasites, selective blocking of the decoration site with human cofilin (HsCOF1) using cytochalasin D increases its severing rate. HsCOF1 may therefore also use a decoration-independent site for filament severing. Thus our data suggest that a second, low affinity actin-binding site may be universally used by ADF/cofilins for actin filament severing. PMID:24371134

  2. A small molecule inhibitor of tropomyosin dissociates actin binding from tropomyosin-directed regulation of actin dynamics

    PubMed Central

    Bonello, Teresa T.; Janco, Miro; Hook, Jeff; Byun, Alex; Appaduray, Mark; Dedova, Irina; Hitchcock-DeGregori, Sarah; Hardeman, Edna C.; Stehn, Justine R.; Böcking, Till; Gunning, Peter W.

    2016-01-01

    The tropomyosin family of proteins form end-to-end polymers along the actin filament. Tumour cells rely on specific tropomyosin-containing actin filament populations for growth and survival. To dissect out the role of tropomyosin in actin filament regulation we use the small molecule TR100 directed against the C terminus of the tropomyosin isoform Tpm3.1. TR100 nullifies the effect of Tpm3.1 on actin depolymerisation but surprisingly Tpm3.1 retains the capacity to bind F-actin in a cooperative manner. In vivo analysis also confirms that, in the presence of TR100, fluorescently tagged Tpm3.1 recovers normally into stress fibers. Assembling end-to-end along the actin filament is thereby not sufficient for tropomyosin to fulfil its function. Rather, regulation of F-actin stability by tropomyosin requires fidelity of information communicated at the barbed end of the actin filament. This distinction has significant implications for perturbing tropomyosin-dependent actin filament function in the context of anti-cancer drug development. PMID:26804624

  3. CASEIN KINASE1-LIKE PROTEIN2 Regulates Actin Filament Stability and Stomatal Closure via Phosphorylation of Actin Depolymerizing Factor.

    PubMed

    Zhao, Shuangshuang; Jiang, Yuxiang; Zhao, Yang; Huang, Shanjin; Yuan, Ming; Zhao, Yanxiu; Guo, Yan

    2016-06-01

    The opening and closing of stomata are crucial for plant photosynthesis and transpiration. Actin filaments undergo dynamic reorganization during stomatal closure, but the underlying mechanism for this cytoskeletal reorganization remains largely unclear. In this study, we identified and characterized Arabidopsis thaliana casein kinase 1-like protein 2 (CKL2), which responds to abscisic acid (ABA) treatment and participates in ABA- and drought-induced stomatal closure. Although CKL2 does not bind to actin filaments directly and has no effect on actin assembly in vitro, it colocalizes with and stabilizes actin filaments in guard cells. Further investigation revealed that CKL2 physically interacts with and phosphorylates actin depolymerizing factor 4 (ADF4) and inhibits its activity in actin filament disassembly. During ABA-induced stomatal closure, deletion of CKL2 in Arabidopsis alters actin reorganization in stomata and renders stomatal closure less sensitive to ABA, whereas deletion of ADF4 impairs the disassembly of actin filaments and causes stomatal closure to be more sensitive to ABA Deletion of ADF4 in the ckl2 mutant partially recues its ABA-insensitive stomatal closure phenotype. Moreover, Arabidopsis ADFs from subclass I are targets of CKL2 in vitro. Thus, our results suggest that CKL2 regulates actin filament reorganization and stomatal closure mainly through phosphorylation of ADF. PMID:27268429

  4. Evidence That an Unconventional Actin Can Provide Essential F-Actin Function and That a Surveillance System Monitors F-Actin Integrity in Chlamydomonas.

    PubMed

    Onishi, Masayuki; Pringle, John R; Cross, Frederick R

    2016-03-01

    Actin is one of the most conserved eukaryotic proteins. It is thought to have multiple essential cellular roles and to function primarily or exclusively as filaments ("F-actin"). Chlamydomonas has been an enigma, because a null mutation (ida5-1) in its single gene for conventional actin does not affect growth. A highly divergent actin gene, NAP1, is upregulated in ida5-1 cells, but it has been unclear whether NAP1 can form filaments or provide actin function. Here, we used the actin-depolymerizing drug latrunculin B (LatB), the F-actin-specific probe Lifeact-Venus, and genetic and molecular methods to resolve these issues. LatB-treated wild-type cells continue to proliferate; they initially lose Lifeact-stained structures but recover them concomitant with upregulation of NAP1. Thirty-nine LatB-sensitive mutants fell into four genes (NAP1 and LAT1-LAT3) in which we identified the causative mutations using a novel combinatorial pool-sequencing strategy. LAT1-LAT3 are required for NAP1 upregulation upon LatB treatment, and ectopic expression of NAP1 largely rescues the LatB sensitivity of the lat1-lat3 mutants, suggesting that the LAT gene products comprise a regulatory hierarchy with NAP1 expression as the major functional output. Selection of LatB-resistant revertants of a nap1 mutant yielded dominant IDA5 mutations that presumably render F-IDA5 resistant to LatB, and nap1 and lat mutations are synthetically lethal with ida5-1 in the absence of LatB. We conclude that both IDA5 and the divergent NAP1 can form filaments and redundantly provide essential F-actin functions and that a novel surveillance system, probably responding to a loss of F-actin, triggers NAP1 expression and perhaps other compensatory responses. PMID:26715672

  5. Actin and Endocytosis in Budding Yeast

    PubMed Central

    Goode, Bruce L.; Eskin, Julian A.; Wendland, Beverly

    2015-01-01

    Endocytosis, the process whereby the plasma membrane invaginates to form vesicles, is essential for bringing many substances into the cell and for membrane turnover. The mechanism driving clathrin-mediated endocytosis (CME) involves > 50 different protein components assembling at a single location on the plasma membrane in a temporally ordered and hierarchal pathway. These proteins perform precisely choreographed steps that promote receptor recognition and clustering, membrane remodeling, and force-generating actin-filament assembly and turnover to drive membrane invagination and vesicle scission. Many critical aspects of the CME mechanism are conserved from yeast to mammals and were first elucidated in yeast, demonstrating that it is a powerful system for studying endocytosis. In this review, we describe our current mechanistic understanding of each step in the process of yeast CME, and the essential roles played by actin polymerization at these sites, while providing a historical perspective of how the landscape has changed since the preceding version of the YeastBook was published 17 years ago (1997). Finally, we discuss the key unresolved issues and where future studies might be headed. PMID:25657349

  6. Encoding Mechano-Memories in Actin Networks

    NASA Astrophysics Data System (ADS)

    Foucard, Louis; Majumdar, Sayantan; Levine, Alex; Gardel, Margaret

    The ability of cells to sense and adapt to external mechanical stimuli is vital to many of its biological functions. A critical question is therefore to understand how mechanosensory mechanisms arise in living matter, with implications in both cell biology and smart materials design. Experimental work has demonstrated that the mechanical properties of semiflexible actin networks in Eukaryotic cells can be modulated (either transiently or irreversibly) via the application of external forces. Previous work has also shown with a combination of numerical simulations and analytic calculations shows that the broken rotational symmetry of the filament orientational distribution in semiflexible networks leads to dramatic changes in the mechanical response. Here we demonstrate with a combination of numerical and analytic calculations that the observed long-lived mechano-memory in the actin networks arise from changes in the nematic order of the constituent filaments. These stress-induced changes in network topology relax slowly under zero stress and can be observed through changes in the nonlinear mechanics. Our results provide a strategy for designing a novel class of materials and demonstrate a new putative mechanism of mechanical sensing in eukaryotic cells.

  7. Actin Foci Adhesion of D. discoideum

    NASA Astrophysics Data System (ADS)

    Flanders, Bret; Paneru, Govind

    2014-03-01

    Amoeboid migration is a fast (10 μm min-1) integrin-independent mode of migration that is important with D. discoideum, leukocytes, and breast cancer cells. It is poorly understood, but depends on the establishment of adhesive contacts to the substrate where the cell transmits traction forces. In pre-aggregative D. discoideum, a model system for learning about amoeboid migration, these adhesive contacts are discrete complexes that are known as actin-foci. They have an area of ~ 0.5 μm2 and a lifetime of ~ 20 s. This talk will present measurements of the adhesive character of actin foci that have been obtained using a submicron force transducer that was designed for this purpose. Results on the rupture stresses and lifetimes of individual acting foci under nano-newton level forces will be described in the context of a general theory for cellular adhesion. This theory depends on, essentially, three cellular properties: the membrane-medium surface tension, the number density of adhesion receptors in the membrane, and the receptor-substrate potential energy surface. Therefore, the use of the transducer to determine the surface tension will be presented, as well.

  8. Actin binding proteins, spermatid transport and spermiation.

    PubMed

    Qian, Xiaojing; Mruk, Dolores D; Cheng, Yan-Ho; Tang, Elizabeth I; Han, Daishu; Lee, Will M; Wong, Elissa W P; Cheng, C Yan

    2014-06-01

    The transport of germ cells across the seminiferous epithelium is composed of a series of cellular events during the epithelial cycle essential to the completion of spermatogenesis. Without the timely transport of spermatids during spermiogenesis, spermatozoa that are transformed from step 19 spermatids in the rat testis fail to reach the luminal edge of the apical compartment and enter the tubule lumen at spermiation, thereby arriving the epididymis for further maturation. Step 19 spermatids and/or sperms that remain in the epithelium beyond stage VIII of the epithelial cycle will be removed by the Sertoli cell via phagocytosis to form phagosomes and be degraded by lysosomes, leading to subfertility and/or infertility. However, the biology of spermatid transport, in particular the final events that lead to spermiation remain elusive. Based on recent data in the field, we critically evaluate the biology of spermiation herein by focusing on the actin binding proteins (ABPs) that regulate the organization of actin microfilaments at the Sertoli-spermatid interface, which is crucial for spermatid transport during this event. The hypothesis we put forth herein also highlights some specific areas of research that can be pursued by investigators in the years to come.

  9. Force Transmission in the Actin Cytoskeleton

    NASA Astrophysics Data System (ADS)

    Gardel, Margaret

    2012-02-01

    The ability of cells to sense and generate mechanical forces is essential to numerous aspects of their physiology, including adhesion, migration, division and differentiation. To a large degree, cellular tension is regulated by the transmission of myosin II-generated forces through the filamentous actin (F-actin) cytoskeleton. While transmission of myosin-generated stresses from the molecular to cellular length scale is well understood in the context of highly organized sarcomeres found in striated muscle, non-muscle and smooth muscle cells contain a wide variety of bundles and networks lacking sarcomeric organization. I will describe the in vitro and in vivo approaches we use to study force transmission in such disordered actomyosin assemblies. Our in vivo results are showing that highly organized stress fibers contribute surprisingly little to the overall extent of cellular tension as compared to disordered actomyosin meshworks. Our in vitro results are demonstrating the mechanisms of symmetry breaking in disordered actomyosin bundles that facilitate the formation of contractile bundles with well-defined ``contractile elements.'' These results provide insight into the self-organization of actomyosin cytoskeleton in non-muscle cells that regulate and maintain cellular tension.

  10. Mechanics of composite actin networks: in vitro and cellular perspectives

    NASA Astrophysics Data System (ADS)

    Upadhyaya, Arpita

    2014-03-01

    Actin filaments and associated actin binding proteins play an essential role in governing the mechanical properties of eukaryotic cells. Even though cells have multiple actin binding proteins (ABPs) that exist simultaneously to maintain the structural and mechanical integrity of the cellular cytoskeleton, how these proteins work together to determine the properties of actin networks is not well understood. The ABP, palladin, is essential for the integrity of cell morphology and movement during development. Palladin coexists with alpha-actinin in stress fibers and focal adhesions and binds to both actin and alpha-actinin. To obtain insight into how mutually interacting actin crosslinking proteins modulate the properties of actin networks, we have characterized the micro-structure and mechanics of actin networks crosslinked with palladin and alpha-actinin. Our studies on composite networks of alpha-actinin/palladin/actin show that palladin and alpha-actinin synergistically determine network viscoelasticity. We have further examined the role of palladin in cellular force generation and mechanosensing. Traction force microscopy revealed that TAFs are sensitive to substrate stiffness as they generate larger forces on substrates of increased stiffness. Contrary to expectations, knocking down palladin increased the forces generated by cells, and also inhibited the ability to sense substrate stiffness for very stiff gels. This was accompanied by significant differences in the actin organization and adhesion dynamics of palladin knock down cells. Perturbation experiments also suggest altered myosin activity in palladin KD cells. Our results suggest that the actin crosslinkers such as palladin and myosin motors coordinate for optimal cell function and to prevent aberrant behavior as in cancer metastasis.

  11. Drebrin Inhibits Cofilin-Induced Severing of F-Actin

    PubMed Central

    Grintsevich, Elena E.; Reisler, Emil

    2015-01-01

    Molecular cross-talk between neuronal drebrin A and cofilin is believed to be a part of the activity-dependent cytoskeleton-modulating pathway in dendritic spines. Impairments in this pathway are implicated also in synaptic dysfunction in Alzheimer’s disease, Down syndrome, epilepsy, and normal aging. However, up to now the molecular interplay between cofilin and drebrin has not been elucidated. TIRF microscopy and solution experiments revealed that full length drebrin A or its actin binding core (Drb1-300) inhibits, but do not abolish cofilin-induced severing of actin filaments. Cosedimentation experiments showed that F-actin can be fully occupied with combination of these two proteins. The dependence of cofilin binding on fractional saturation of actin filaments with drebrin suggests direct competition between these two proteins for F-actin binding. This implies that cofilin and drebrin can either overcome or reverse the allosteric changes in F-actin induced by the competitor’s binding. The ability of cofilin to displace drebrin from actin filaments is pH dependent and is facilitated at acidic pH (6.8). Pre-steady state kinetic experiments reveal that both binding and dissociation of drebrin to/from actin filaments is faster than that reported for cooperative binding of cofilin. We found, that drebrin displacement by cofilin is greatly inhibited when actin severing is abolished, which might be linked to the cooperativity of drebrin binding to actin filaments. Our results contribute to molecular understanding of the competitive interactions of drebrin and cofilin with actin filaments. PMID:25047716

  12. Computational Analysis of Viscoelastic Properties of Crosslinked Actin Networks

    PubMed Central

    Kim, Taeyoon; Hwang, Wonmuk; Lee, Hyungsuk; Kamm, Roger D.

    2009-01-01

    Mechanical force plays an important role in the physiology of eukaryotic cells whose dominant structural constituent is the actin cytoskeleton composed mainly of actin and actin crosslinking proteins (ACPs). Thus, knowledge of rheological properties of actin networks is crucial for understanding the mechanics and processes of cells. We used Brownian dynamics simulations to study the viscoelasticity of crosslinked actin networks. Two methods were employed, bulk rheology and segment-tracking rheology, where the former measures the stress in response to an applied shear strain, and the latter analyzes thermal fluctuations of individual actin segments of the network. It was demonstrated that the storage shear modulus (G′) increases more by the addition of ACPs that form orthogonal crosslinks than by those that form parallel bundles. In networks with orthogonal crosslinks, as crosslink density increases, the power law exponent of G′ as a function of the oscillation frequency decreases from 0.75, which reflects the transverse thermal motion of actin filaments, to near zero at low frequency. Under increasing prestrain, the network becomes more elastic, and three regimes of behavior are observed, each dominated by different mechanisms: bending of actin filaments, bending of ACPs, and at the highest prestrain tested (55%), stretching of actin filaments and ACPs. In the last case, only a small portion of actin filaments connected via highly stressed ACPs support the strain. We thus introduce the concept of a ‘supportive framework,’ as a subset of the full network, which is responsible for high elasticity. Notably, entropic effects due to thermal fluctuations appear to be important only at relatively low prestrains and when the average crosslinking distance is comparable to or greater than the persistence length of the filament. Taken together, our results suggest that viscoelasticity of the actin network is attributable to different mechanisms depending on the amount

  13. Computational analysis of viscoelastic properties of crosslinked actin networks.

    PubMed

    Kim, Taeyoon; Hwang, Wonmuk; Lee, Hyungsuk; Kamm, Roger D

    2009-07-01

    Mechanical force plays an important role in the physiology of eukaryotic cells whose dominant structural constituent is the actin cytoskeleton composed mainly of actin and actin crosslinking proteins (ACPs). Thus, knowledge of rheological properties of actin networks is crucial for understanding the mechanics and processes of cells. We used Brownian dynamics simulations to study the viscoelasticity of crosslinked actin networks. Two methods were employed, bulk rheology and segment-tracking rheology, where the former measures the stress in response to an applied shear strain, and the latter analyzes thermal fluctuations of individual actin segments of the network. It was demonstrated that the storage shear modulus (G') increases more by the addition of ACPs that form orthogonal crosslinks than by those that form parallel bundles. In networks with orthogonal crosslinks, as crosslink density increases, the power law exponent of G' as a function of the oscillation frequency decreases from 0.75, which reflects the transverse thermal motion of actin filaments, to near zero at low frequency. Under increasing prestrain, the network becomes more elastic, and three regimes of behavior are observed, each dominated by different mechanisms: bending of actin filaments, bending of ACPs, and at the highest prestrain tested (55%), stretching of actin filaments and ACPs. In the last case, only a small portion of actin filaments connected via highly stressed ACPs support the strain. We thus introduce the concept of a 'supportive framework,' as a subset of the full network, which is responsible for high elasticity. Notably, entropic effects due to thermal fluctuations appear to be important only at relatively low prestrains and when the average crosslinking distance is comparable to or greater than the persistence length of the filament. Taken together, our results suggest that viscoelasticity of the actin network is attributable to different mechanisms depending on the amount of

  14. Targeting the actin cytoskeleton: selective antitumor action via trapping PKCɛ

    PubMed Central

    Foerster, F; Braig, S; Moser, C; Kubisch, R; Busse, J; Wagner, E; Schmoeckel, E; Mayr, D; Schmitt, S; Huettel, S; Zischka, H; Mueller, R; Vollmar, A M

    2014-01-01

    Targeting the actin cytoskeleton (CSK) of cancer cells offers a valuable strategy in cancer therapy. There are a number of natural compounds that interfere with the actin CSK, but the mode of their cytotoxic action and, moreover, their tumor-specific mechanisms are quite elusive. We used the myxobacterial compound Chondramide as a tool to first elucidate the mechanisms of cytotoxicity of actin targeting in breast cancer cells (MCF7, MDA-MB-231). Chondramide inhibits cellular actin filament dynamics shown by a fluorescence-based analysis (fluorescence recovery after photobleaching (FRAP)) and leads to apoptosis characterized by phosphatidylserine exposure, release of cytochrome C from mitochondria and finally activation of caspases. Chondramide enhances the occurrence of mitochondrial permeability transition (MPT) by affecting known MPT modulators: Hexokinase II bound to the voltage-dependent anion channel (VDAC) translocated from the outer mitochondrial membrane to the cytosol and the proapoptotic protein Bad were recruited to the mitochondria. Importantly, protein kinase C-ɛ (PKCɛ), a prosurvival kinase possessing an actin-binding site and known to regulate the hexokinase/VDAC interaction as well as Bad phosphorylation was identified as the link between actin CSK and apoptosis induction. PKCɛ, which was found overexpressed in breast cancer cells, accumulated in actin bundles induced by Chondramide and lost its activity. Our second goal was to characterize the potential tumor-specific action of actin-binding agents. As the nontumor breast epithelial cell line MCF-10A in fact shows resistance to Chondramide-induced apoptosis and notably express low level of PKCɛ, we suggest that trapping PKCɛ via Chondramide-induced actin hyperpolymerization displays tumor cell specificity. Our work provides a link between targeting the ubiquitously occurring actin CSK and selective inhibition of pro-tumorigenic PKCɛ, thus setting the stage for actin-stabilizing agents as

  15. Bending Flexibility of Actin Filaments during Motor-Induced Sliding

    PubMed Central

    Vikhorev, Petr G.; Vikhoreva, Natalia N.; Månsson, Alf

    2008-01-01

    Muscle contraction and other forms of cell motility occur as a result of cyclic interactions between myosin molecules and actin filaments. Force generation is generally attributed to ATP-driven structural changes in myosin, whereas a passive role is ascribed to actin. However, some results challenge this view, predicting structural changes in actin during motor activity, e.g., when the actin filaments slide on a myosin-coated surface in vitro. Here, we analyzed statistical properties of the sliding filament paths, allowing us to detect changes of this type. It is interesting to note that evidence for substantial structural changes that led to increased bending flexibility of the filaments was found in phalloidin-stabilized, but not in phalloidin-free, actin filaments. The results are in accordance with the idea that a high-flexibility structural state of actin is a prerequisite for force production, but not the idea that a low-to-high flexibility transition of the actin filament should be an important component of the force-generating step per se. Finally, our data challenge the general view that phalloidin-stabilized filaments behave as native actin filaments in their interaction with myosin. This has important implications, since phalloidin stabilization is a routine procedure in most studies of actomyosin function. PMID:18835897

  16. Lateral Membrane Diffusion Modulated by a Minimal Actin Cortex

    PubMed Central

    Heinemann, Fabian; Vogel, Sven K.; Schwille, Petra

    2013-01-01

    Diffusion of lipids and proteins within the cell membrane is essential for numerous membrane-dependent processes including signaling and molecular interactions. It is assumed that the membrane-associated cytoskeleton modulates lateral diffusion. Here, we use a minimal actin cortex to directly study proposed effects of an actin meshwork on the diffusion in a well-defined system. The lateral diffusion of a lipid and a protein probe at varying densities of membrane-bound actin was characterized by fluorescence correlation spectroscopy (FCS). A clear correlation of actin density and reduction in mobility was observed for both the lipid and the protein probe. At high actin densities, the effect on the protein probe was ∼3.5-fold stronger compared to the lipid. Moreover, addition of myosin filaments, which contract the actin mesh, allowed switching between fast and slow diffusion in the minimal system. Spot variation FCS was in accordance with a model of fast microscopic diffusion and slower macroscopic diffusion. Complementing Monte Carlo simulations support the analysis of the experimental FCS data. Our results suggest a stronger interaction of the actin mesh with the larger protein probe compared to the lipid. This might point toward a mechanism where cortical actin controls membrane diffusion in a strong size-dependent manner. PMID:23561523

  17. Filament Assembly by Spire: Key Residues and Concerted Actin Binding

    PubMed Central

    Rasson, Amy S.; Bois, Justin S.; Pham, Duy Stephen L.; Yoo, Haneul; Quinlan, Margot E.

    2014-01-01

    The most recently identified class of actin nucleators, WASp Homology domain 2 (WH2) – nucleators, use tandem repeats of monomeric actin-binding WH2 domains to facilitate actin nucleation. WH2 domains are involved in a wide variety of actin regulatory activities. Structurally, they are expected to clash with interprotomer contacts within the actin filament. Thus, the discovery of their role in nucleation was surprising. Here we use Drosophila Spire (Spir) as a model system to investigate both how tandem WH2 domains can nucleate actin and what differentiates nucleating WH2-containing proteins from their non-nucleating counterparts. We found that the third WH2 domain in Spir (Spir-C or Sc), plays a unique role. In the context of a short nucleation construct (containing only two WH2 domains), placement of Sc in the N-terminal position was required for the most potent nucleation. We found that the native organization of the WH2 domains with respect to each other is necessary for binding to actin with positive cooperativity. We identified two residues within Sc that are critical for its activity. Using this information we were able to convert a weak synthetic nucleator into one with activity equal to a native Spir construct. Lastly, we found evidence that Sc binds actin filaments, in addition to monomers. PMID:25234086

  18. Actin Interacts with Dengue Virus 2 and 4 Envelope Proteins.

    PubMed

    Jitoboam, Kunlakanya; Phaonakrop, Narumon; Libsittikul, Sirikwan; Thepparit, Chutima; Roytrakul, Sittiruk; Smith, Duncan R

    2016-01-01

    Dengue virus (DENV) remains a significant public health problem in many tropical and sub-tropical countries worldwide. The DENV envelope (E) protein is the major antigenic determinant and the protein that mediates receptor binding and endosomal fusion. In contrast to some other DENV proteins, relatively few cellular interacting proteins have been identified. To address this issue a co-immuoprecipitation strategy was employed. The predominant co-immunoprecipitating proteins identified were actin and actin related proteins, however the results suggested that actin was the only bona fide interacting partner. Actin was shown to interact with the E protein of DENV 2 and 4, and the interaction between actin and DENV E protein was shown to occur in a truncated DENV consisting of only domains I and II. Actin was shown to decrease during infection, but this was not associated with a decrease in gene transcription. Actin-related proteins also showed a decrease in expression during infection that was not transcriptionally regulated. Cytoskeletal reorganization was not observed during infection, suggesting that the interaction between actin and E protein has a cell type specific component. PMID:27010925

  19. Auxin Stimulates Its Own Transport by Shaping Actin Filaments1

    PubMed Central

    Nick, Peter; Han, Min-Jung; An, Gyeunhung

    2009-01-01

    The directional transport of the plant hormone auxin has been identified as central element of axis formation and patterning in plants. This directionality of transport depends on gradients, across the cell, of auxin-efflux carriers that continuously cycle between plasma membrane and intracellular compartments. This cycling has been proposed to depend on actin filaments. However, the role of actin for the polarity of auxin transport has been disputed. The organization of actin, in turn, has been shown to be under control of auxin. By overexpression of the actin-binding protein talin, we have generated transgenic rice (Oryza sativa) lines, where actin filaments are bundled to variable extent and, in consequence, display a reduced dynamics. We show that this bundling of actin filaments correlates with impaired gravitropism and reduced longitudinal transport of auxin. We can restore a normal actin configuration by addition of exogenous auxins and restore gravitropism as well as polar auxin transport. This rescue is mediated by indole-3-acetic acid and 1-naphthyl acetic acid but not by 2,4-dichlorophenoxyacetic acid. We interpret these findings in the context of a self-referring regulatory circuit between polar auxin transport and actin organization. This circuit might contribute to the self-amplification of auxin transport that is a central element in current models of auxin-dependent patterning. PMID:19633235

  20. Accelerators, Brakes, and Gears of Actin Dynamics in Dendritic Spines

    PubMed Central

    Pontrello, Crystal G.; Ethell, Iryna M.

    2010-01-01

    Dendritic spines are actin-rich structures that accommodate the postsynaptic sites of most excitatory synapses in the brain. Although dendritic spines form and mature as synaptic connections develop, they remain plastic even in the adult brain, where they can rapidly grow, change, or collapse in response to normal physiological changes in synaptic activity that underlie learning and memory. Pathological stimuli can adversely affect dendritic spine shape and number, and this is seen in neurodegenerative disorders and some forms of mental retardation and autism as well. Many of the molecular signals that control these changes in dendritic spines act through the regulation of filamentous actin (F-actin), some through direct interaction with actin, and others via downstream effectors. For example, cortactin, cofilin, and gelsolin are actin-binding proteins that directly regulate actin dynamics in dendritic spines. Activities of these proteins are precisely regulated by intracellular signaling events that control their phosphorylation state and localization. In this review, we discuss how actin-regulating proteins maintain the balance between F-actin assembly and disassembly that is needed to stabilize mature dendritic spines, and how changes in their activities may lead to rapid remodeling of dendritic spines. PMID:20463852

  1. Actin puts the squeeze on Drosophila glue secretion.

    PubMed

    Merrifield, Christien J

    2016-02-01

    An actin filament coat promotes cargo expulsion from large exocytosing vesicles, but the mechanisms of coat formation and force generation have been poorly characterized. Elegant imaging studies of the Drosophila melanogaster salivary gland now reveal how actin and myosin are recruited, and show that myosin II forms a contractile 'cage' that facilitates exocytosis.

  2. Filament assembly by Spire: key residues and concerted actin binding.

    PubMed

    Rasson, Amy S; Bois, Justin S; Pham, Duy Stephen L; Yoo, Haneul; Quinlan, Margot E

    2015-02-27

    The most recently identified class of actin nucleators, WASp homology domain 2 (WH2) nucleators, use tandem repeats of monomeric actin-binding WH2 domains to facilitate actin nucleation. WH2 domains are involved in a wide variety of actin regulatory activities. Structurally, they are expected to clash with interprotomer contacts within the actin filament. Thus, the discovery of their role in nucleation was surprising. Here we use Drosophila Spire (Spir) as a model system to investigate both how tandem WH2 domains can nucleate actin and what differentiates nucleating WH2-containing proteins from their non-nucleating counterparts. We found that the third WH2 domain in Spir (Spir-C or SC) plays a unique role. In the context of a short nucleation construct (containing only two WH2 domains), placement of SC in the N-terminal position was required for the most potent nucleation. We found that the native organization of the WH2 domains with respect to each other is necessary for binding to actin with positive cooperativity. We identified two residues within SC that are critical for its activity. Using this information, we were able to convert a weak synthetic nucleator into one with activity equal to a native Spir construct. Lastly, we found evidence that SC binds actin filaments, in addition to monomers.

  3. Actin Interacts with Dengue Virus 2 and 4 Envelope Proteins

    PubMed Central

    Jitoboam, Kunlakanya; Phaonakrop, Narumon; Libsittikul, Sirikwan; Thepparit, Chutima; Roytrakul, Sittiruk; Smith, Duncan R.

    2016-01-01

    Dengue virus (DENV) remains a significant public health problem in many tropical and sub-tropical countries worldwide. The DENV envelope (E) protein is the major antigenic determinant and the protein that mediates receptor binding and endosomal fusion. In contrast to some other DENV proteins, relatively few cellular interacting proteins have been identified. To address this issue a co-immuoprecipitation strategy was employed. The predominant co-immunoprecipitating proteins identified were actin and actin related proteins, however the results suggested that actin was the only bona fide interacting partner. Actin was shown to interact with the E protein of DENV 2 and 4, and the interaction between actin and DENV E protein was shown to occur in a truncated DENV consisting of only domains I and II. Actin was shown to decrease during infection, but this was not associated with a decrease in gene transcription. Actin-related proteins also showed a decrease in expression during infection that was not transcriptionally regulated. Cytoskeletal reorganization was not observed during infection, suggesting that the interaction between actin and E protein has a cell type specific component. PMID:27010925

  4. Actin filament nucleation and elongation factors--structure-function relationships.

    PubMed

    Dominguez, Roberto

    2009-01-01

    The spontaneous and unregulated polymerization of actin filaments is inhibited in cells by actin monomer-binding proteins such as profilin and Tbeta4. Eukaryotic cells and certain pathogens use filament nucleators to stabilize actin polymerization nuclei, whose formation is rate-limiting. Known filament nucleators include the Arp2/3 complex and its large family of nucleation promoting factors (NPFs), formins, Spire, Cobl, VopL/VopF, TARP and Lmod. These molecules control the time and location for polymerization, and additionally influence the structures of the actin networks that they generate. Filament nucleators are generally unrelated, but with the exception of formins they all use the WASP-Homology 2 domain (WH2 or W), a small and versatile actin-binding motif, for interaction with actin. A common architecture, found in Spire, Cobl and VopL/VopF, consists of tandem W domains that bind three to four actin subunits to form a nucleus. Structural considerations suggest that NPFs-Arp2/3 complex can also be viewed as a specialized form of tandem W-based nucleator. Formins are unique in that they use the formin-homology 2 (FH2) domain for interaction with actin and promote not only nucleation, but also processive barbed end elongation. In contrast, the elongation function among W-based nucleators has been "outsourced" to a dedicated family of proteins, Eva/VASP, which are related to WASP-family NPFs.

  5. Deafness and espin-actin self-organization in stereocilia

    NASA Astrophysics Data System (ADS)

    Wong, Gerard C. L.

    2009-03-01

    Espins are F-actin-bundling proteins associated with large parallel actin bundles found in hair cell stereocilia in the ear, as well as brush border microvilli and Sertoli cell junctions. We examine actin bundle structures formed by different wild-type espin isoforms, fragments, and naturally-occurring human espin mutants linked to deafness and/or vestibular dysfunction. The espin-actin bundle structure consisted of a hexagonal arrangement of parallel actin filaments in a non-native twist state. We delineate the structural consequences caused by mutations in espin's actin-bundling module. For espin mutation with a severely damaged actin-bundling module, which are implicated in deafness in mice and humans, oriented nematic-like actin filament structures, which strongly impinges on bundle mechanical stiffness. Finally, we examine what makes espin different, via a comparative study of bundles formed by espin and those formed by fascin, a prototypical bundling protein found in functionally different regions of the cell, such as filopodia.

  6. G-actin guides p53 nuclear transport: potential contribution of monomeric actin in altered localization of mutant p53.

    PubMed

    Saha, Taniya; Guha, Deblina; Manna, Argha; Panda, Abir Kumar; Bhat, Jyotsna; Chatterjee, Subhrangsu; Sa, Gaurisankar

    2016-01-01

    p53 preserves genomic integrity by restricting anomaly at the gene level. Till date, limited information is available for cytosol to nuclear shuttling of p53; except microtubule-based trafficking route, which utilizes minus-end directed motor dynein. The present study suggests that monomeric actin (G-actin) guides p53 traffic towards the nucleus. Histidine-tag pull-down assay using purified p53(1-393)-His and G-actin confirms direct physical association between p53 and monomeric G-actin. Co-immunoprecipitation data supports the same. Confocal imaging explores intense perinuclear colocalization between p53 and G-actin. To address atomistic details of the complex, constraint-based docked model of p53:G-actin complex was generated based on crystal structures. MD simulation reveals that p53 DNA-binding domain arrests very well the G-actin protein. Docking benchmark studies have been carried out for a known crystal structure, 1YCS (complex between p53DBD and BP2), which validates the docking protocol we adopted. Co-immunoprecipitation study using "hot-spot" p53 mutants suggested reduced G-actin association with cancer-associated p53 conformational mutants (R175H and R249S). Considering these findings, we hypothesized that point mutation in p53 structure, which diminishes p53:G-actin complexation results in mutant p53 altered subcellular localization. Our model suggests p53Arg249 form polar-contact with Arg357 of G-actin, which upon mutation, destabilizes p53:G-actin interaction and results in cytoplasmic retention of p53R249S. PMID:27601274

  7. G-actin guides p53 nuclear transport: potential contribution of monomeric actin in altered localization of mutant p53

    PubMed Central

    Saha, Taniya; Guha, Deblina; Manna, Argha; Panda, Abir Kumar; Bhat, Jyotsna; Chatterjee, Subhrangsu; Sa, Gaurisankar

    2016-01-01

    p53 preserves genomic integrity by restricting anomaly at the gene level. Till date, limited information is available for cytosol to nuclear shuttling of p53; except microtubule-based trafficking route, which utilizes minus-end directed motor dynein. The present study suggests that monomeric actin (G-actin) guides p53 traffic towards the nucleus. Histidine-tag pull-down assay using purified p53(1–393)-His and G-actin confirms direct physical association between p53 and monomeric G-actin. Co-immunoprecipitation data supports the same. Confocal imaging explores intense perinuclear colocalization between p53 and G-actin. To address atomistic details of the complex, constraint-based docked model of p53:G-actin complex was generated based on crystal structures. MD simulation reveals that p53 DNA-binding domain arrests very well the G-actin protein. Docking benchmark studies have been carried out for a known crystal structure, 1YCS (complex between p53DBD and BP2), which validates the docking protocol we adopted. Co-immunoprecipitation study using “hot-spot” p53 mutants suggested reduced G-actin association with cancer-associated p53 conformational mutants (R175H and R249S). Considering these findings, we hypothesized that point mutation in p53 structure, which diminishes p53:G-actin complexation results in mutant p53 altered subcellular localization. Our model suggests p53Arg249 form polar-contact with Arg357 of G-actin, which upon mutation, destabilizes p53:G-actin interaction and results in cytoplasmic retention of p53R249S. PMID:27601274

  8. Actin-Based Feedback Circuits in Cell Migration and Endocytosis

    NASA Astrophysics Data System (ADS)

    Wang, Xinxin

    In this thesis, we study the switch and pulse functions of actin during two important cellular processes, cell migration and endocytosis. Actin is an abundant protein that can polymerize to form a dendritic network. The actin network can exert force to push or bend the cell membrane. During cell migration, the actin network behaves like a switch, assembling mostly at one end or at the other end. The end with the majority of the actin network is the leading edge, following which the cell can persistently move in the same direction. The other end, with the minority of the actin network, is the trailing edge, which is dragged by the cell as it moves forward. When subjected to large fluctuations or external stimuli, the leading edge and the trailing edge can interchange and change the direction of motion, like a motion switch. Our model of the actin network in a cell reveals that mechanical force is crucial for forming the motion switch. We find a transition from single state symmetric behavior to switch behavior, when tuning parameters such as the force. The model is studied by both stochastic simulations, and a set of rate equations that are consistent with the simulations. Endocytosis is a process by which cells engulf extracellular substances and recycle the cell membrane. In yeast cells, the actin network is transiently needed to overcome the pressure difference across the cell membrane caused by turgor pressure. The actin network behaves like a pulse, which assembles and then disassembles within about 30 seconds. Using a stochastic model, we reproduce the pulse behaviors of the actin network and one of its regulatory proteins, Las17. The model matches green fluorescence protein (GFP) experiments for wild-type cells. The model also predicts some phenotypes that modify or diminish the pulse behavior. The phenotypes are verified with both experiments performed at Washington University and with other groups' experiments. We find that several feedback mechanisms are

  9. Interior decoration: tropomyosin in actin dynamics and cell migration.

    PubMed

    Lees, Justin G; Bach, Cuc T T; O'Neill, Geraldine M

    2011-01-01

    Cell migration and invasion requires the precise temporal and spatial orchestration of a variety of biological processes. Filaments of polymerized actin are critical players in these diverse processes, including the regulation of cell anchorage points (both cell-cell and cell-extracellular matrix), the uptake and delivery of molecules via endocytic pathways and the generation of force for both membrane protrusion and retraction. How the actin filaments are specialized for each of these discrete functions is yet to be comprehensively elucidated. The cytoskeletal tropomyosins are a family of actin associating proteins that form head-to-tail polymers which lay in the major groove of polymerized actin filaments. In the present review we summarize the emerging isoform-specific functions of tropomyosins in cell migration and invasion and discuss their potential roles in the specialization of actin filaments for the diverse cellular processes that together regulate cell migration and invasion.

  10. Actin-associated Proteins in the Pathogenesis of Podocyte Injury

    PubMed Central

    He, Fang-Fang; Chen, Shan; Su, Hua; Meng, Xian-Fang; Zhang, Chun

    2013-01-01

    Podocytes have a complex cellular architecture with interdigitating processes maintained by a precise organization of actin filaments. The actin-based foot processes of podocytes and the interposed slit diaphragm form the final barrier to proteinuria. The function of podocytes is largely based on the maintenance of the normal foot process structure with actin cytoskeleton. Cytoskeletal dynamics play important roles during normal podocyte development, in maintenance of the healthy glomerular filtration barrier, and in the pathogenesis of glomerular diseases. In this review, we focused on recent findings on the mechanisms of organization and reorganization of these actin-related molecules in the pathogenesis of podocyte injury and potential therapeutics targeting the regulation of actin cytoskeleton in podocytopathies. PMID:24396279

  11. Side-binding proteins modulate actin filament dynamics.

    PubMed

    Crevenna, Alvaro H; Arciniega, Marcelino; Dupont, Aurélie; Mizuno, Naoko; Kowalska, Kaja; Lange, Oliver F; Wedlich-Söldner, Roland; Lamb, Don C

    2015-01-01

    Actin filament dynamics govern many key physiological processes from cell motility to tissue morphogenesis. A central feature of actin dynamics is the capacity of filaments to polymerize and depolymerize at their ends in response to cellular conditions. It is currently thought that filament kinetics can be described by a single rate constant for each end. In this study, using direct visualization of single actin filament elongation, we show that actin polymerization kinetics at both filament ends are strongly influenced by the binding of proteins to the lateral filament surface. We also show that the pointed-end has a non-elongating state that dominates the observed filament kinetic asymmetry. Estimates of flexibility as well as effects on fragmentation and growth suggest that the observed kinetic diversity arises from structural alteration. Tuning elongation kinetics by exploiting the malleability of the filament structure may be a ubiquitous mechanism to generate a rich variety of cellular actin dynamics. PMID:25706231

  12. Role of Actin Polymerization in Cell Locomotion: Molecules and Models

    PubMed Central

    Bearer, E. L.

    2015-01-01

    Actin filaments forming at the anterior margin of a migrating cell are essential for the formation of filopodia, lamellipodia, and pseudopodia, the “feet” that the cell extends before it. These structures in turn are required for cell locomotion. Yet the molecular nature of the “nucleator” that seeds the polymerization of actin at the leading edge is unknown. Recent advances, including video microscopy of actin dynamics, discovery of proteins unique to the leading edge such as ponticulin, the Mab 2E4 antigen, and ABP 120, and novel experimental models of actin polymerization such as the actin-based movements of intracellular parasites, promise to shed light on this problem in the near future. PMID:8323743

  13. Structural Modeling and Molecular Dynamics Simulation of the Actin Filament

    SciTech Connect

    Splettstoesser, Thomas; Holmes, Kenneth; Noe, Frank; Smith, Jeremy C

    2011-01-01

    Actin is a major structural protein of the eukaryotic cytoskeleton and enables cell motility. Here, we present a model of the actin filament (F-actin) that not only incorporates the global structure of the recently published model by Oda et al. but also conserves internal stereochemistry. A comparison is made using molecular dynamics simulation of the model with other recent F-actin models. A number of structural determents such as the protomer propeller angle, the number of hydrogen bonds, and the structural variation among the protomers are analyzed. The MD comparison is found to reflect the evolution in quality of actin models over the last 6 years. In addition, simulations of the model are carried out in states with both ADP or ATP bound and local hydrogen-bonding differences characterized.

  14. Actinic prurigo of the lip: Two case reports

    PubMed Central

    Miranda, Ana MO; Ferrari, Thiago M; Werneck, Juliana T; Junior, Arley Silva; Cunha, Karin S; Dias, Eliane P

    2014-01-01

    Actinic prurigo is a photodermatosis that can affect the skin, conjunctiva and lips. It is caused by an abnormal reaction to sunlight and is more common in high-altitude living people, mainly in indigenous descendants. The diagnosis of actinic prurigo can be challenging, mainly when lip lesions are the only manifestation, which is not a common clinical presentation. The aim of this article is to report two cases of actinic prurigo showing only lip lesions. The patients were Afro-American and were unaware of possible Indian ancestry. Clinical exam, photographs, videoroscopy examination and biopsy were performed, and the diagnosis of actinic prurigo was established. Topical corticosteroid and lip balm with ultraviolet protection were prescribed with excellent results. The relevance of this report is to show that although some patients may not demonstrate the classical clinical presentation of actinic prurigo, the associated clinical and histological exams are determinants for the correct diagnosis and successful treatment of this disease. PMID:25133153

  15. How actin initiates the motor activity of Myosin.

    PubMed

    Llinas, Paola; Isabet, Tatiana; Song, Lin; Ropars, Virginie; Zong, Bin; Benisty, Hannah; Sirigu, Serena; Morris, Carl; Kikuti, Carlos; Safer, Dan; Sweeney, H Lee; Houdusse, Anne

    2015-05-26

    Fundamental to cellular processes are directional movements driven by molecular motors. A common theme for these and other molecular machines driven by ATP is that controlled release of hydrolysis products is essential for using the chemical energy efficiently. Mechanochemical transduction by myosin motors on actin is coupled to unknown structural changes that result in the sequential release of inorganic phosphate (Pi) and MgADP. We present here a myosin structure possessing an actin-binding interface and a tunnel (back door) that creates an escape route for Pi with a minimal rotation of the myosin lever arm that drives movements. We propose that this state represents the beginning of the powerstroke on actin and that Pi translocation from the nucleotide pocket triggered by actin binding initiates myosin force generation. This elucidates how actin initiates force generation and movement and may represent a strategy common to many molecular machines.

  16. IFT88 influences chondrocyte actin organization and biomechanics

    PubMed Central

    Wang, Z.; Wann, A.K.T.; Thompson, C.L.; Hassen, A.; Wang, W.; Knight, M.M.

    2016-01-01

    Summary Objectives Primary cilia are microtubule based organelles which control a variety of signalling pathways important in cartilage development, health and disease. This study examines the role of the intraflagellar transport (IFT) protein, IFT88, in regulating fundamental actin organisation and mechanics in articular chondrocytes. Methods The study used an established chondrocyte cell line with and without hypomorphic mutation of IFT88 (IFT88orpk). Confocal microscopy was used to quantify F-actin and myosin IIB organisation. Viscoelastic cell and actin cortex mechanics were determined using micropipette aspiration with actin dynamics visualised in live cells transfected with LifeACT-GFP. Results IFT88orpk cells exhibited a significant increase in acto-myosin stress fibre organisation relative to wild-type (WT) cells in monolayer and an altered response to cytochalasin D. Rounded IFT88orpk cells cultured in suspension exhibited reduced cortical actin expression with reduced cellular equilibrium modulus. Micropipette aspiration resulted in reduced membrane bleb formation in IFT88orpk cells. Following membrane blebbing, IFT88orpk cells exhibited slower reformation of the actin cortex. IFT88orpk cells showed increased actin deformability and reduced cortical tension confirming that IFT regulates actin cortex mechanics. The reduced cortical tension is also consistent with the reduced bleb formation. Conclusions This study demonstrates for the first time that the ciliary protein IFT88 regulates fundamental actin organisation and the stiffness of the actin cortex leading to alterations in cell deformation, mechanical properties and blebbing in an IFT88 chondrocyte cell line. This adds to the growing understanding of the role of primary cilia and IFT in regulating cartilage biology. PMID:26493329

  17. Force Generation, Polymerization Dynamics and Nucleation of Actin Filaments

    NASA Astrophysics Data System (ADS)

    Wang, Ruizhe

    We study force generation and actin filament dynamics using stochastic and deterministic methods. First, we treat force generation of bundled actin filaments by polymerization via molecular-level stochastic simulations. In the widely-used Brownian Ratchet model, actin filaments grow freely whenever the tip-obstacle gap created by thermal fluctuation exceeds the monomer size. We name this model the Perfect Brownian Ratchet (PBR) model. In the PBR model, actin monomer diffusion is treated implicitly. We perform a series of simulations based on the PBR, in which obstacle motion is treated explicitly; in most previous studies, obstacle motion has been treated implicitly. We find that the cooperativity of filaments is generally weak in the PBR model, meaning that more filaments would grow more slowly given the same force per filament. Closed-form formulas are also developed, which match the simulation results. These portable and accurate formulas provide guidance for experiments and upper and lower bounds for theoretical analyses. We also studied a variation of the PBR, called the Diffusing Brownian Ratchet (DBR) model, in which both actin monomer and obstacle diffusion are treated explicitly. We find that the growth rate of multiple filaments is even lower, compared with that in PBR. This finding challenges the widely-accepted PBR assumption and suggests that pushing the study of actin dynamics down to the sub-nanometer level yields new insights. We subsequently used a rate equation approach to model the effect of local depletion of actin monomers on the nucleation of actin filaments on biomimetic beads, and how the effect is regulated by capping protein (CP). We find that near the bead surface, a higher CP concentration increases local actin concentration, which leads to an enhanced activities of actin filaments' nucleation. Our model analysis matches the experimental results and lends support to an important but undervalued hypothesis proposed by Carlier and

  18. A Structural Basis for Regulation of Actin Polymerization by Pectenotoxins

    PubMed Central

    Allingham, John S.; Miles, Christopher O.; Rayment, Ivan

    2007-01-01

    Pectenotoxins (PTXs) are polyether macrolides found in certain dinoflagellates, sponges and shellfish, and have been associated with diarrhetic shellfish poisoning. In addition to their in vivo toxicity, some PTXs are potently cytotoxic in human cancer cell lines. Recent studies have demonstrated that disruption of the actin cytoskeleton may be a key function of these compounds, although no clarification their mechanism of action at a molecular level was available. We have obtained an X-ray crystal structure of PTX-2 bound to actin which, in combination with analyses of the effect of PTX-2 on purified actin filament dynamics, provides a molecular explanation for its effects on actin. PTX-2 formed a 1:1 complex with actin and engaged a novel site between subdomains 1 and 3. Based on models of the actin filament, PTX binding would disrupt key lateral contacts between the PTX-bound actin monomer and the lower lateral actin monomer within the filament, thereby capping the barbed-end. The location of this binding position within the interior of the filament indicates that it may not be accessible once polymerization has occurred, a hypothesis supported by our observation that PTX-2 caused filament capping without inducing filament severing. This mode of action is unique, as other actin filament destabilizing toxins appear to exclusively disrupt longitudinal monomer contacts allowing many of them to sever filaments in addition to capping them. Examination of the PTX-binding site on actin provides a rationalization for the structure–activity relationships observed in vivo and in vitro, and may provide a basis for predicting toxicity of PTX analogues. PMID:17599353

  19. A dynamic formin-dependent deep F-actin network in axons

    PubMed Central

    Ganguly, Archan; Tang, Yong; Wang, Lina; Ladt, Kelsey; Loi, Jonathan; Dargent, Bénédicte; Leterrier, Christophe

    2015-01-01

    Although actin at neuronal growth cones is well-studied, much less is known about actin organization and dynamics along axon shafts and presynaptic boutons. Using probes that selectively label filamentous-actin (F-actin), we found focal “actin hotspots” along axons—spaced ∼3–4 µm apart—where actin undergoes continuous assembly/disassembly. These foci are a nidus for vigorous actin polymerization, generating long filaments spurting bidirectionally along axons—a phenomenon we call “actin trails.” Super-resolution microscopy reveals intra-axonal deep actin filaments in addition to the subplasmalemmal “actin rings” described recently. F-actin hotspots colocalize with stationary axonal endosomes, and blocking vesicle transport diminishes the actin trails, suggesting mechanistic links between vesicles and F-actin kinetics. Actin trails are formin—but not Arp2/3—dependent and help enrich actin at presynaptic boutons. Finally, formin inhibition dramatically disrupts synaptic recycling. Collectively, available data suggest a two-tier F-actin organization in axons, with stable “actin rings” providing mechanical support to the plasma membrane and dynamic "actin trails" generating a flexible cytoskeletal network with putative physiological roles. PMID:26216902

  20. SelR/MsrB Reverses Mical-mediated Oxidation of Actin to Regulate F-actin Dynamics

    PubMed Central

    Hung, Ruei-Jiun; Spaeth, Christopher S.; Yesilyurt, Hunkar Gizem; Terman, Jonathan R.

    2014-01-01

    Actin's polymerization properties are dramatically altered by oxidation of its conserved methionine (Met)-44 residue. Mediating this effect is a specific oxidation-reduction (Redox) enzyme, Mical, that works with Semaphorin repulsive guidance cues and selectively oxidizes Met-44. We now find that this actin regulatory process is reversible. Employing a genetic approach, we identified a specific methionine sulfoxide reductase enzyme SelR that opposes Mical Redox activity and Semaphorin/Plexin repulsion to direct multiple actin-dependent cellular behaviors in vivo. SelR specifically catalyzes the reduction of the R-isomer of methionine sulfoxide (methionine-R-sulfoxide) to methionine, and we found that SelR directly reduced Mical-oxidized actin, restoring its normal polymerization properties. These results indicate that Mical oxidizes actin stereo-specifically to generate actin Met-44-R-sulfoxide (actinMet(R)O-44) – and they also implicate the interconversion of specific Met/Met(R)O residues as a precise means to modulate protein function. Our results therefore uncover a specific reversible Redox actin regulatory system that controls cell and developmental biology. PMID:24212093

  1. Triggering Actin Comets Versus Membrane Ruffles: Distinctive Effects of Phosphoinositides on Actin Reorganization

    PubMed Central

    Ueno, Tasuku; Falkenburger, Björn H.; Pohlmeyer, Christopher; Inoue, Takanari

    2012-01-01

    A limited set of phosphoinositide membrane lipids regulate diverse cellular functions including proliferation, differentiation, and migration. We developed two techniques based on rapamycin-induced protein dimerization to rapidly change the concentration of plasma membrane phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. First, we increased PI(4,5)P2 synthesis from phosphatidylinositol 4-phosphate [PI(4)P] using a membrane recruitable form of PI(4)P 5-kinase, and found that COS-7, HeLa, and HEK293 cells formed bundles of motile actin filaments known as actin comets. In contrast, a second technique that increased the concentration of PI(4,5)P2 without consuming PI(4)P induced membrane ruffles. These distinct phenotypes were mediated by dynamin-mediated vesicular trafficking and mutually inhibitory crosstalk between the small guanosine triphosphatases Rac and RhoA. Our results indicate that the effect of PI(4,5)P2 on actin reorganization depends on the abundance of other phosphoinositides, such as PI(4)P. Thus, combinatorial regulation of phosphoinositide concentrations may contribute to the diversity of phosphoinositide functions. PMID:22169478

  2. 75 FR 3641 - Television Broadcasting Services; Anchorage, AK

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-01-22

    ... From the Federal Register Online via the Government Publishing Office FEDERAL COMMUNICATIONS COMMISSION 47 CFR Part 73 Television Broadcasting Services; Anchorage, AK AGENCY: Federal Communications... broadcasting. 0 For the reasons discussed in the preamble, the Federal Communications Commission amends 47...

  3. 78 FR 55772 - Alaska Disaster Number AK-00028

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-09-11

    ... From the Federal Register Online via the Government Publishing Office ] SMALL BUSINESS ADMINISTRATION Alaska Disaster Number AK-00028 AGENCY: U.S. Small Business Administration. ACTION: Amendment 1... information in the original declaration remains unchanged. (Catalog of Federal Domestic Assistance...

  4. Viral Replication Protein Inhibits Cellular Cofilin Actin Depolymerization Factor to Regulate the Actin Network and Promote Viral Replicase Assembly

    PubMed Central

    Kovalev, Nikolay; de Castro Martín, Isabel Fernández; Barajas, Daniel; Risco, Cristina; Nagy, Peter D.

    2016-01-01

    RNA viruses exploit host cells by co-opting host factors and lipids and escaping host antiviral responses. Previous genome-wide screens with Tomato bushy stunt virus (TBSV) in the model host yeast have identified 18 cellular genes that are part of the actin network. In this paper, we show that the p33 viral replication factor interacts with the cellular cofilin (Cof1p), which is an actin depolymerization factor. Using temperature-sensitive (ts) Cof1p or actin (Act1p) mutants at a semi-permissive temperature, we find an increased level of TBSV RNA accumulation in yeast cells and elevated in vitro activity of the tombusvirus replicase. We show that the large p33 containing replication organelle-like structures are located in the close vicinity of actin patches in yeast cells or around actin cable hubs in infected plant cells. Therefore, the actin filaments could be involved in VRC assembly and the formation of large viral replication compartments containing many individual VRCs. Moreover, we show that the actin network affects the recruitment of viral and cellular components, including oxysterol binding proteins and VAP proteins to form membrane contact sites for efficient transfer of sterols to the sites of replication. Altogether, the emerging picture is that TBSV, via direct interaction between the p33 replication protein and Cof1p, controls cofilin activities to obstruct the dynamic actin network that leads to efficient subversion of cellular factors for pro-viral functions. In summary, the discovery that TBSV interacts with cellular cofilin and blocks the severing of existing filaments and the formation of new actin filaments in infected cells opens a new window to unravel the way by which viruses could subvert/co-opt cellular proteins and lipids. By regulating the functions of cofilin and the actin network, which are central nodes in cellular pathways, viruses could gain supremacy in subversion of cellular factors for pro-viral functions. PMID:26863541

  5. Actin-cytoskeleton dynamics in non-monotonic cell spreading

    PubMed Central

    Heinrich, Doris; Youssef, Simon; Schroth-Diez, Britta; Engel, Ulrike; Aydin, Daniel; Blümmel, Jacques; Spatz, Joachim P

    2008-01-01

    The spreading of motile cells on a substrate surface is accompanied by reorganization of their actin network. We show that spreading in the highly motile cells of Dictyostelium is non-monotonic, and thus differs from the passage of spreading cells through a regular series of stages. Quantification of the gain and loss of contact area revealed fluctuating forces of protrusion and retraction that dominate the interaction of Dictyostelium cells with a substrate. The molecular basis of these fluctuations is elucidated by dual-fluorescence labeling of filamentous actin together with proteins that highlight specific activities in the actin system. Front-to-tail polarity is established by the sorting out of myosin-II from regions where dense actin assemblies are accumulating. Myosin-IB identifies protruding front regions, and the Arp2/3 complex localizes to lamellipodia protruded from the fronts. Coronin is used as a sensitive indicator of actin disassembly to visualize the delicate balance of polymerization and depolymerization in spreading cells. Short-lived actin patches that co-localize with clathrin suggest that membrane internalization occurs even when the substrate-attached cell surface expands. We conclude that non-monotonic cell spreading is characterized by spatiotemporal patterns formed by motor proteins together with regulatory proteins that either promote or terminate actin polymerization on the scale of seconds. PMID:19262103

  6. Drebrin attenuates the interaction between actin and myosin-V.

    PubMed

    Ishikawa, Ryoki; Katoh, Kaoru; Takahashi, Ayumi; Xie, Ce; Oseki, Koushi; Watanabe, Michitoshi; Igarashi, Michihiro; Nakamura, Akio; Kohama, Kazuhiro

    2007-07-27

    Drebrin-A is an actin-binding protein localized in the dendritic spines of mature neurons, and has been suggested to affect spine morphology [K. Hayashi, T. Shirao, Change in the shape of dendritic spines caused by overexpression of drebrin in cultured cortical neurons, J. Neurosci. 19 (1999) 3918-3925]. However, no biochemical analysis of drebrin-A has yet been reported. In this study, we purified drebrin-A using a bacterial expression system, and characterized it in vitro. Drebrin-A bound to actin filaments with a stoichiometry of one drebrin molecule to 5-6 actin molecules. Furthermore, drebrin-A decreased the Mg-ATPase activity of myosin V. In vitro motility assay revealed that the attachment of F-actin to glass surface coated with myosin-V was decreased by drebrin-A, but once F-actin attached to the surface, the sliding speed of F-actin was unaffected by the presence of drebrin A. These findings suggest that drebrin-A may affect spine dynamics, vesicle transport, and other myosin-V-driven motility in neurons through attenuating the interaction between actin and myosin-V.

  7. Simulation of the effect of confinement in actin ring formation

    NASA Astrophysics Data System (ADS)

    Adeli Koudehi, Maral; Vavylonis, Dimitrios; Haosu Tang Team; Dimitrios Vavylonis Team

    Actin filaments are vital for different network structures in living cells. During cytokinesis, they form a contractile ring containing myosin motor proteins and actin filament cross-linkers to separate one cell into two cells. Recent experimental studies have quantified the bundle, ring, and network structures that form when actin filaments polymerize in confined environments in vitro, in the presence of varying concentrations of cross-linkers. In this study, we performed numerical simulations to investigate the effect of actin spherical confinement and cross-linking in ring formation. We used a spring-bead model and Brownian dynamics to simulate semiflexible actin filaments that polymerize in a confining sphere with a rate proportional to the monomer concentration. Applying the model for different size of the confining spheres shows that the probability of ring formation decreases by increasing the radius (at fixed initial monomer concentration), in agreement with prior experimental data. We describe the effect of persistence length, orientation-dependent cross-linking, and initial actin monomer concentration. Simulations show that equilibrium configurations can be reached through zipping and unzipping of actin filaments in bundles and transient ring formation.

  8. Quantitative fluorescent speckle microscopy (QFSM) to measure actin dynamics.

    PubMed

    Mendoza, Michelle C; Besson, Sebastien; Danuser, Gaudenz

    2012-10-01

    Quantitative fluorescent speckle microscopy (QFSM) is a live-cell imaging method to analyze the dynamics of macromolecular assemblies with high spatial and temporal resolution. Its greatest successes were in the analysis of actin filament and adhesion dynamics in the context of cell migration and microtubule dynamics in interphase and the meiotic/mitotic spindle. Here, focus is on the former application to illustrate the procedures of FSM imaging and the computational image processing that extracts quantitative information from these experiments. QFSM is advantageous over other methods because it measures the movement and turnover kinetics of the actin filament (F-actin) network in living cells across the entire field of view. Experiments begin with the microinjection of fluorophore-labeled actin into cells, which generate a low ratio of fluorescently labeled to endogenously unlabeled actin monomers. Spinning disk confocal or wide-field imaging then visualizes fluorophore clusters (two to eight actin monomers) within the assembled F-actin network as speckles. QFSM software identifies and computationally tracks and utilizes the location, appearance, and disappearance of speckles to derive network flows and maps of the rate of filament assembly and disassembly. PMID:23042526

  9. Concentration profiles of actin-binding molecules in lamellipodia

    NASA Astrophysics Data System (ADS)

    Falcke, Martin

    2016-04-01

    Motile cells form lamellipodia in the direction of motion, which are flat membrane protrusions containing an actin filament network. The network flows rearward relative to the leading edge of the lamellipodium due to actin polymerization at the front. Thus, actin binding molecules are subject to transport towards the rear of the cell in the bound state and diffuse freely in the unbound state. We analyze this reaction-diffusion-advection process with respect to the concentration profiles of these species and provide an analytic approximation for them. Network flow may cause a depletion zone of actin binding molecules close to the leading edge. The existence of such zone depends on the free molecule concentration in the cell body, on the ratio of the diffusion length to the distance bound molecules travel rearward with the flow before dissociating, and the ratio of the diffusion length to the width of the region with network flow and actin binding. Our calculations suggest the existence of depletion zones for the F-actin cross-linkers filamin and α-actinin in fish keratocytes (and other cell types), which is in line with the small elastic moduli of the F-actin network close to the leading edge found in measurements of the force motile cells are able to exert.

  10. Interconnection between actin cytoskeleton and plant defense signaling.

    PubMed

    Janda, Martin; Matoušková, Jindřiška; Burketová, Lenka; Valentová, Olga

    2014-01-01

    Actin cytoskeleton is the fundamental structural component of eukaryotic cells. It has a role in numerous elementary cellular processes such as reproduction, development and also in response to abiotic and biotic stimuli. Remarkably, the role of actin cytoskeleton in plant response to pathogens is getting to be under magnifying glass. Based on microscopic studies, most of the data showed, that actin plays an important role in formation of physiological barrier in the site of infection. Actin dynamics is involved in the transport of antimicrobial compounds and cell wall fortifying components (e.g. callose) to the site of infection. Also the role in PTI (pathogen triggered immunity) and ETI (effector triggered immunity) was recently indicated. On the other hand much less is known about the transcriptome reprogramming upon changes in actin dynamics. Our recently published results showed that drugs inhibiting actin polymerization (latrunculin B, cytochalasin E) cause the induction of genes which are involved in salicylic acid (SA) signaling pathway. In this addendum we would like to highlight in more details current state of knowledge concerning the involvement of actin dynamics in plant defense signaling.

  11. The centrosome is an actin-organizing center

    PubMed Central

    Farina, Francesca; Gaillard, Jérémie; Guérin, Christophe; Couté, Yohann; Sillibourne, James; Blanchoin, Laurent; Théry, Manuel

    2016-01-01

    Microtubules and actin filaments are the two main cytoskeleton networks supporting intracellular architecture and cell polarity. The centrosome nucleates and anchors microtubules and is therefore considered to be the main microtubule-organizing center. However, recurring, yet unexplained, observations have pointed towards a connection between the centrosome and actin filaments. Here we have used isolated centrosomes to demonstrate that the centrosome can directly promote actin filament assembly. A cloud of centrosome-associated actin filaments could be identified in living cells as well. Actin-filament nucleation at the centrosome was mediated by the nucleation promoting factor WASH in combination with the Arp2/3 complex. Pericentriolar material 1 (PCM1) appeared to modulate the centrosomal actin network by regulating Arp2/3 complex and WASH recruitment to the centrosome. Hence our results reveal an additional facet of the centrosome as an intracellular organizer and provide mechanistic insights into how the centrosome can function as an actin filament-organizing center. PMID:26655833

  12. Symmetry breaking in actin gels - Implications for cellular motility

    NASA Astrophysics Data System (ADS)

    John, Karin; Peyla, Philippe; Misbah, Chaouqi

    2007-03-01

    The physical origin of cell motility is not fully understood. Recently minimal model systems have shown, that polymerizing actin itself can produce a motile force, without the help of motor proteins. Pathogens like Shigella or Listeria use actin to propel themselves forward in their host cell. The same process can be mimicked with polystyrene beads covered with the activating protein ActA, which reside in a solution containing actin monomers. ActA induces the growth of an actin gel at the bead surface. Initially the gel grows symmetrically around the bead until a critical size is reached. Subsequently one observes a symmetry breaking and the gel starts to grow asymmetrically around the bead developing a tail of actin at one side. This symmetry breaking is accompanied by a directed movement of the bead, with the actin tail trailing behind the bead. Force generation relies on the combination of two properties: growth and elasticity of the actin gel. We study this phenomenon theoretically within the framework of a linear elasticity theory and linear flux-force relationships for the evolution of an elastic gel around a hard sphere. Conditions for a parity symmetry breaking are identified analytically and illustrated numerically with the help of a phasefield model.

  13. Phosphorylation of platelet actin-binding protein during platelet activation

    SciTech Connect

    Carroll, R.C.; Gerrard, J.M.

    1982-03-01

    In this study we have followed the 32P-labeling of actin-binding protein as a function of platelet activation. Utilizing polyacrylamide-sodium dodecyl sulfate gel electrophoresis to resolve total platelet protein samples, we found 2 to 3-fold labeling increases in actin-binding protein 30 to 60 sec after thrombin stimulation. Somewhat larger increases were observed for 40,000 and 20,000 apparent molecular weight peptides. The actin-binding protein was identified on the gels by coelectrophoresis with purified actin-binding protein, its presence in cytoskeletal cores prepared by detergent extraction of activated 32P-labeled platelets, and by direct immunoprecipitation with antibodies against guinea pig vas deferens filamin (actin-binding protein). In addition, these cytoskeletal cores indicated that the 32P-labeled actin-binding protein was closely associated with the activated platelet's cytoskeleton. Following the 32P-labeling of actin-binding protein over an 8-min time course revealed that in aggregating platelet samples rapid dephosphorylation to almost initial levels occurred between 3 and 5 min. A similar curve was obtained for the 20,000 apparent molecular weight peptide. However, rapid dephosphorylation was not observed if platelet aggregation was prevented by chelating external calcium or by using thrombasthenic platelets lacking the aggregation response. Thus, cell-cell contact would seem to be crucial in initiating the rapid dephosphorylation response.

  14. Genomic study of polyhydroxyalkanoates producing Aeromonas hydrophila 4AK4.

    PubMed

    Gao, Xue; Jian, Jia; Li, Wen-Jie; Yang, Yu-Cheng; Shen, Xiao-Wen; Sun, Zhi-Rong; Wu, Qiong; Chen, Guo-Qiang

    2013-10-01

    The complete genome of Gram-negative Aeromonas hydrophila 4AK4 that has been used for industrial production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) was sequenced and annotated. Its chromosome is 4,527,993 bp in size encoding 4,272 genes, including 28 rRNA genes and 104 tRNA genes. Comparative analysis indicated that genome of A. hydrophila 4AK4 was similar to that of the A. hydrophila ATCC 7966(T), an intensively studied aeromonad for its pathogenicity related to its genomic information. Genes possibly coming from other species or even other genus were identified in A. hydrophila 4AK4. A large number of putative virulent genes were predicted. However, a cytotonic enterotoxin (Ast) is absent in A. hydrophila 4AK4, allowing the industrial strain to be different from other A. hydrophila strains, indicating possible reduced virulence of strain 4AK4, which is very important for industrial fermentation. Genes involved in polyhydroxyalkanoate (PHA) metabolism were predicted and analyzed. The resulting genomic information is useful for improved production of PHA via metabolic engineering of A. hydrophila 4AK4.

  15. Role of actin in auxin transport and transduction of gravity

    NASA Astrophysics Data System (ADS)

    Hu, S.; Basu, S.; Brady, S.; Muday, G.

    Transport of the plant hormone auxin is polar and the direction of the hormone movement appears to be controlled by asymmetric distribution of auxin transport protein complexes. Changes in the direction of auxin transport are believed to drive asymmetric growth in response to changes in the gravity vector. To test the possibility that asymmetric distribution of the auxin transport protein complex is mediated by attachment to the actin cytoskeleton, a variety of experimental approaches have been used. The most direct demonstration of the role of the actin cytoskeleton in localization of the protein complex is the ability of one protein in this complex to bind to affinity columns containing actin filaments. Additionally, treatments of plant tissues with drugs that fragment the actin c toskeleton reducey polar transport. In order to explore this actin interaction and the affect of gravity on auxin transport and developmental polarity, embryos of the brown alga, Fucus have been examined. Fucus zygotes are initially symmetrical, but develop asymmetry in response to environmental gradients, with light gradients being the best- characterized signal. Gravity will polarize these embryos and gravity-induced polarity is randomized by clinorotation. Auxin transport also appears necessary for environmental controls of polarity, since auxin efflux inhibitors perturb both photo- and gravity-polarization at a very discrete temporal window within six hours after fertilization. The actin cytoskeleton has previously been shown to reorganize after fertilization of Fucus embryos leading to formation of an actin patch at the site of polar outgrowth. These actin patches still form in Fucus embryos treated with auxin efflux inhibitors, yet the position of these patches is randomized. Together, these results suggest that there are connections between the actin cytoskeleton, auxin transport, and gravity oriented growth and development. (Supported by NASA Grant: NAG2-1203)

  16. Analysis of actinic flux profiles measured from an ozonesonde balloon

    NASA Astrophysics Data System (ADS)

    Wang, P.; Allaart, M.; Knap, W. H.; Stammes, P.

    2015-04-01

    A green light sensor has been developed at KNMI to measure actinic flux profiles using an ozonesonde balloon. In total, 63 launches with ascending and descending profiles were performed between 2006 and 2010. The measured uncalibrated actinic flux profiles are analysed using the Doubling-Adding KNMI (DAK) radiative transfer model. Values of the cloud optical thickness (COT) along the flight track were taken from the Spinning Enhanced Visible and Infrared Imager (SEVIRI) Cloud Physical Properties (CPP) product. The impact of clouds on the actinic flux profile is evaluated on the basis of the cloud modification factor (CMF) at the cloud top and cloud base, which is the ratio between the actinic fluxes for cloudy and clear-sky scenes. The impact of clouds on the actinic flux is clearly detected: the largest enhancement occurs at the cloud top due to multiple scattering. The actinic flux decreases almost linearly from cloud top to cloud base. Above the cloud top the actinic flux also increases compared to clear-sky scenes. We find that clouds can increase the actinic flux to 2.3 times the clear-sky value at cloud top and decrease it to about 0.05 at cloud base. The relationship between CMF and COT agrees well with DAK simulations, except for a few outliers. Good agreement is found between the DAK-simulated actinic flux profiles and the observations for single-layer clouds in fully overcast scenes. The instrument is suitable for operational balloon measurements because of its simplicity and low cost. It is worth further developing the instrument and launching it together with atmospheric chemistry composition sensors.

  17. Proteolytic cleavage of actin within the DNase-I-binding loop changes the conformation of F-actin and its sensitivity to myosin binding.

    PubMed

    Borovikov, Y S; Moraczewska, J; Khoroshev, M I; Strzelecka-Gołaszewska, H

    2000-03-16

    Effects of subtilisin cleavage of actin between residues 47 and 48 on the conformation of F-actin and on its changes occurring upon binding of myosin subfragment-1 (S1) were investigated by measuring polarized fluorescence from rhodamine-phalloidin- or 1, 5-IAEDANS-labeled actin filaments reconstructed from intact or subtilisin-cleaved actin in myosin-free muscle fibers (ghost fibers). In separate experiments, polarized fluorescence from 1, 5-IAEDANS-labeled S1 bound to non-labeled actin filaments in ghost fibers was measured. The measurements revealed differences between the filaments of cleaved and intact actin in the orientation of rhodamine probe on the rhodamine-phalloidin-labeled filaments, orientation and mobility of the C-terminus of actin, filament flexibility, and orientation and mobility of the myosin heads bound to F-actin. The changes in the filament flexibility and orientation of the actin-bound fluorophores produced by S1 binding to actin in the absence of ATP were substantially diminished by subtilisin cleavage of actin. The results suggest that loop 38-52 plays an important role, not only in maintaining the F-actin structure, but also in the conformational transitions in actin accompanying the strong binding of the myosin heads that may be essential for the generation of force and movement during actin-myosin interaction.

  18. Actin purification from a gel of rat brain extracts.

    PubMed

    Levilliers, N; Peron-Renner, M; Coffe, G; Pudles, J

    1984-01-01

    Actin, 99% pure, has been recovered from rat brain with a high yield (greater than 15 mg/100 g brain). We have shown that: 1. a low ionic strength extract from rat brain tissue is capable of giving rise to a gel; 2. actin is the main gel component and its proportion is one order of magnitude higher than in the original extract; 3. actin can be isolated from this extract by a three-step procedure involving gelation, dissociation of the gel in 0.6 M KCl, followed by one or two depolymerization-polymerization cycles. PMID:6529588

  19. Actinic reticuloid imitating Sézary syndrome.

    PubMed

    Pacheco, David; Fraga, Ana; Travassos, Ana Rita; Antunes, Joana; Freitas, Joaao; Soares de Almeida, Luis; Filipe, Paulo

    2012-09-01

    Actinic reticuloid is a rare chronic idiopathic photosensitive dermatosis belonging to the spectrum of chronic actinic dermatitis and may be mistaken for cutaneous T-cell lymphoma. We report the case of an erythrodermic patient, initially diagnosed with Sézary syndrome, treated with chlorambucil and prednisolone. Later on, a photobiological study demonstrated photosensitivity to UVB, UVA, and visible light. The clinical picture, histological findings resembling lymphoma, and the results of the photobiological study established the diagnosis of actinic reticuloid. Currently the patient is avoiding sun exposure and being medicated with azathioprine and prednisolone, and is showing sustained improvement. PMID:23267873

  20. Acanthamoeba castellanii: identification and distribution of actin cytoskeleton.

    PubMed

    González-Robles, Arturo; Castañón, Guadalupe; Hernández-Ramírez, Verónica Ivonne; Salazar-Villatoro, Lizbeth; González-Lázaro, Mónica; Omaña-Molina, Maritza; Talamás-Rohana, Patricia; Martínez-Palomo, Adolfo

    2008-07-01

    The presence of the cytoskeleton of Acanthamoeba castellanii was observed by means of cryo-electronmicroscopy and immunofluorescence techniques. This structure is formed largely by fibers and networks of actin located mainly in cytoplasmic locomotion structures as lamellipodia and as well as in various endocytic structures. In addition, the comparison between total actin content in whole extracts among different amoebae was made. The molecular weight of actin in A. castellanii was 44 kDa, and 45 kDa for Naegleria fowleri and Entamoeba histolytica.

  1. Revamping AK-Ashland gas cleaning system

    SciTech Connect

    Brandes, H.; Koerbel, R.; Haberkamp, K.; Keeton, S.

    1995-07-01

    AK Steel`s (formerly Armco) BOF shop was using a static precipitator for the primary collection. The system was designed for full combustion in the gas collecting hoods. No secondary dust collection was in place. A detailed study on alternative solutions led to a completely different system in 1990, and an order was awarded to Mannesmann Demag Corp. (MDC) in Dec. 1990. The new gas collection system is using suppressed combustion with the capability to collect Co at a later stage. The gas cleaning uses the Mannesmann Demag Baumco scrubber with a venturi throat for gas flow control. All auxiliary components, water treatment plant, electric substations and sludge handling were designed and supplied by MDC. The secondary dust collection covers the hot metal and scrap charging into the BOF`s, reladling, desulfurization and deslagging by a pulse jet baghouse. All emission limits set by the EPA and guaranteed by MDC have been met by the systems installed.

  2. Curved trajectories of actin-based motility in two dimensions

    NASA Astrophysics Data System (ADS)

    Wen, Fu-Lai; Leung, Kwan-tai; Chen, Hsuan-Yi

    2012-05-01

    Recent experiments have reported fascinating geometrical trajectories for actin-based motility of bacteria Listeria monocytogenes and functionalized beads. To understand the physical mechanism for these trajectories, we constructed a phenomenological model to study the motion of an actin-propelled disk in two dimensions. In our model, the force and actin density on the surface of the disk are influenced by the translation and rotation of the disk, which in turn is induced by the asymmetric distributions of those densities. We show that this feedback can destabilize a straight trajectory, leading to circular, S-shape and other geometrical trajectories observed in the experiments through bifurcations in the distributions of the force and actin density. The relation between our model and the models for self-propelled deformable particles is emphasized and discussed.

  3. Nanosecond electric pulses trigger actin responses in plant cells

    SciTech Connect

    Berghoefer, Thomas; Eing, Christian; Flickinger, Bianca; Hohenberger, Petra; Wegner, Lars H.; Frey, Wolfgang; Nick, Peter

    2009-09-25

    We have analyzed the cellular effects of nanosecond pulsed electrical fields on plant cells using fluorescently tagged marker lines in the tobacco cell line BY-2 and confocal laser scanning microscopy. We observe a disintegration of the cytoskeleton in the cell cortex, followed by contraction of actin filaments towards the nucleus, and disintegration of the nuclear envelope. These responses are accompanied by irreversible permeabilization of the plasma membrane manifest as uptake of Trypan Blue. By pretreatment with the actin-stabilizing drug phalloidin, the detachment of transvacuolar actin from the cell periphery can be suppressed, and this treatment can also suppress the irreversible perforation of the plasma membrane. We discuss these findings in terms of a model, where nanosecond pulsed electric fields trigger actin responses that are key events in the plant-specific form of programmed cell death.

  4. Tracing myoblast fusion in Drosophila embryos by fluorescent actin probes.

    PubMed

    Haralalka, Shruti; Abmayr, Susan M

    2015-01-01

    Myoblast fusion in the Drosophila embryo is a highly elaborate process that is initiated by Founder Cells and Fusion-Competent Myoblasts (FCMs). It occurs through an asymmetric event in which actin foci assemble in the FCMs at points of cell-cell contact and direct the formation of membrane protrusions that drive fusion. Herein, we describe the approach that we have used to image in living embryos the highly dynamic actin foci and actin-rich projections that precede myoblast fusion. We discuss resources currently available for imaging actin and myogenesis, and our experience with these resources if available. This technical report is not intended to be comprehensive on providing instruction on standard microscopy practices or software utilization. However, we discuss microscope parameters that we have used in data collection, and our experience with image processing tools in data analysis.

  5. Cortactin Branches Out: Roles in Regulating Protrusive Actin Dynamics

    PubMed Central

    Ammer, Amanda Gatesman; Weed, Scott A.

    2008-01-01

    Since its discovery in the early 1990’s, cortactin has emerged as a key signaling protein in many cellular processes, including cell adhesion, migration, endocytosis, and tumor invasion. While the list of cellular functions influenced by cortactin grows, the ability of cortactin to interact with and alter the cortical actin network is central to its role in regulating these processes. Recently, several advances have been made in our understanding of the interaction between actin and cortactin, providing insight into how these two proteins work together to provide a framework for normal and altered cellular function. This review examines how regulation of cortactin through post-translational modifications and interactions with multiple binding partners elicits changes in cortical actin cytoskeletal organization, impacting the regulation and formation of actin-rich motility structures. PMID:18615630

  6. Measuring actin dynamics during phagocytosis using photo-switchable fluorescence

    NASA Astrophysics Data System (ADS)

    Kovari, Daniel T.; Curtis, Jennifer E.

    2013-03-01

    Phagocytosis has traditionally been investigated in terms of the relevant biochemical signaling pathways. However, a growing number of studies investigating the physical aspects of phagocytosis have demonstrated that several distinct forces are exerted throughout particle ingestion. We use variations on FRAP (Fluorescence Recovery After Photobleaching) in combination with photo-switchable fluorescent protein to investigate actin dynamics as a phagocyte attempts to engulf its prey. The goal of our actin studies are to determine the recruitment and polymerization rate of actin in the forming phagosome and whether an organized contractile actin ring is present and responsible for phagosome closure, as proposed in the literature. These experiments are ongoing and contribute to our long term effort of developing a physics based model of phagocytosis.

  7. Induction of anti-actin drug resistance in Tetrahymena.

    PubMed

    Zackroff, Robert V; Hufnagel, Linda A

    2002-01-01

    Both cytochalasin D and latrunculin B reversibly inhibited Tetrahymena phagocytosis at concentrations similar to those effective in mammalian systems, even though ciliate actins are known to be highly divergent from mammalian actins. Overnight exposure to relatively low (0.25 microM) concentrations of latrunculin B induced resistance in Tetrahymena to the inhibitory effects of that drug, as well as cross-resistance to cytochalasin D. However, much higher (> 30 microM) concentrations of cytochalasin D were required for induction of cross-resistance to latrunculin B. Anti-actin drug resistance in Tetrahymena may involve a general multidrug resistance mechanism and/or specific feedback regulation of F-actin assembly and stability.

  8. Stable high capacity, F-actin affinity column

    SciTech Connect

    Luna, E.J.; Wang, Y.L.; Voss, E.W. Jr.; Branton, D.; Taylor, D.L.

    1982-11-10

    A high capacity F-actin affinity matrix is constructed by binding fluorescyl-actin to rabbit anti-fluorescein IgG that is covalently bound to Sepharose 4B. When stabilized with phalloidin, the actin remains associated with the Sepharose beads during repeated washes, activates the ATPase activity of myosin subfragment 1, and specifically binds /sup 125/I-heavy meromyosin and /sup 125/I-tropomyosin. The associations between the F-actin-binding proteins are monitored both by affinity chromatography and by a rapid, low speed sedimentation assay. Anti-fluorescein IgG-Sepharose should be generally useful as a matrix for the immobilization of proteins containing accessible, covalently bound fluorescein groups.

  9. A model actin comet tail disassembling by severing

    PubMed Central

    Michalski, P J; Carlsson, A E

    2011-01-01

    We use a numerical simulation to model an actin comet tail as it grows from the surface of a small object (a bead) and disassembles by severing. We explore the dependence of macroscopic properties such as the local tail radius and tail length on several controllable properties, namely, the bead diameter, the bead velocity, the severing rate per unit length, and the actin gel mesh size. The model predicts an F-actin density with an initial exponential decay followed by an abrupt decay at the edge of the tail, and predicts that the comet tail diameter is constant along the length of the tail. The simulation results are used to fit a formula relating the comet tail length to the control parameters, and it is proposed that this formula offers a means to extract quantitative information on the actin gel mesh size and severing kinetics from simple macroscopic measurements. PMID:21566272

  10. Computational defect review for actinic mask inspections

    NASA Astrophysics Data System (ADS)

    Morgan, Paul; Rost, Daniel; Price, Daniel; Corcoran, Noel; Satake, Masaki; Hu, Peter; Peng, Danping; Yonenaga, Dean; Tolani, Vikram

    2013-04-01

    As optical lithography continues to extend into low-k1 regime, resolution of mask patterns continues to diminish. The limitation of 1.35 NA posed by water-based lithography has led to the application of various resolution enhancement techniques (RET), for example, use of strong phase-shifting masks, aggressive OPC and sub-resolution assist features, customized illuminators, etc. The adoption of these RET techniques combined with the requirements to detect even smaller defects on masks due to increasing MEEF, poses considerable challenges for a mask inspection engineer. Inspecting masks under their actinic-aerial image conditions would detect defects that are more likely to print under those exposure conditions. However, this also makes reviewing such defects in their low-contrast aerial images very challenging. On the other hand, inspecting masks under higher resolution inspection optics would allow for better viewing of defects post-inspection. However, such inspections generally would also detect many more defects, including printable and nuisance, thereby making it difficult to judge which are of real concern for printability on wafer. Often, an inspection engineer may choose to use Aerial and/or high resolution inspection modes depending on where in the process flow the mask is and the specific device-layer characteristics of the mask. Hence, a comprehensive approach is needed in handling defects both post-aerial and post-high resolution inspections. This analysis system is designed for the Applied Materials Aera™ mask inspection platform, all data reported was collected using the Aera.

  11. The role of actin networks in cellular mechanosensing

    NASA Astrophysics Data System (ADS)

    Azatov, Mikheil

    Physical processes play an important role in many biological phenomena, such as wound healing, organ development, and tumor metastasis. During these processes, cells constantly interact with and adapt to their environment by exerting forces to mechanically probe the features of their surroundings and generating appropriate biochemical responses. The mechanisms underlying how cells sense the physical properties of their environment are not well understood. In this thesis, I present my studies to investigate cellular responses to the stiffness and topography of the environment. In order to sense the physical properties of their environment, cells dynamically reorganize the structure of their actin cytoskeleton, a dynamic network of biopolymers, altering the shape and spatial distribution of protein assemblies. Several observations suggest that proteins that crosslink actin filaments may play an important role in cellular mechanosensitivity. Palladin is an actin-crosslinking protein that is found in the lamellar actin network, stress fibers and focal adhesions, cellular structures that are critical for mechanosensing of the physical environment. By virtue of its close interactions with these structures in the cell, palladin may play an important role in cell mechanics. However, the role of actin crosslinkers in general, and palladin in particular, in cellular force generation and mechanosensing is not well known. I have investigated the role of palladin in regulating the plasticity of the actin cytoskeleton and cellular force generation in response to alterations in substrate stiffness. I have shown that the expression levels of palladin modulate the forces exerted by cells and their ability to sense substrate stiffness. Perturbation experiments also suggest that palladin levels in cells altered myosin motor activity. These results suggest that the actin crosslinkers, such as palladin, and myosin motors coordinate for optimal cell function and to prevent aberrant

  12. The core and conserved role of MAL is homeostatic regulation of actin levels.

    PubMed

    Salvany, Lara; Muller, Julius; Guccione, Ernesto; Rørth, Pernille

    2014-05-15

    The transcription cofactor MAL is regulated by free actin levels and thus by actin dynamics. MAL, together with its DNA-binding partner, SRF, is required for invasive cell migration and in experimental metastasis. Although MAL/SRF has many targets, we provide genetic evidence in both Drosophila and human cellular models that actin is the key target that must be regulated by MAL/SRF for invasive cell migration. By regulating MAL/SRF activity, actin protein feeds back on production of actin mRNA to ensure sufficient supply of actin. This constitutes a dedicated homeostatic feedback system that provides a foundation for cellular actin dynamics.

  13. Sequential activation of alpha-actin genes during avian cardiogenesis: vascular smooth muscle alpha-actin gene transcripts mark the onset of cardiomyocyte differentiation

    PubMed Central

    1988-01-01

    The expression of cytoplasmic beta-actin and cardiac, skeletal, and smooth muscle alpha-actins during early avian cardiogenesis was analyzed by in situ hybridization with mRNA-specific single-stranded DNA probes. The cytoplasmic beta-actin gene was ubiquitously expressed in the early chicken embryo. In contrast, the alpha-actin genes were sequentially activated in avian cardiac tissue during the early stages of heart tube formation. The accumulation of large quantities of smooth muscle alpha-actin transcripts in epimyocardial cells preceded the expression of the sarcomeric alpha-actin genes. The accumulation of skeletal alpha-actin mRNAs in the developing heart lagged behind that of cardiac alpha-actin by several embryonic stages. At Hamburger- Hamilton stage 12, the smooth muscle alpha-actin gene was selectively down-regulated in the heart such that only the conus, which subsequently participates in the formation of the vascular trunks, continued to express this gene. This modulation in smooth muscle alpha- actin gene expression correlated with the beginning of coexpression of sarcomeric alpha-actin transcripts in the epimyocardium and the onset of circulation in the embryo. The specific expression of the vascular smooth muscle alpha-actin gene marks the onset of differentiation of cardiac cells and represents the first demonstration of coexpression of both smooth muscle and striated alpha-actin genes within myogenic cells. PMID:3204121

  14. 78 FR 34609 - Proposed Modification of Class D and E Airspace; Kenai, AK

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-06-10

    ... airspace, Class E surface airspace and Class E airspace designated as an extension to Class D surface area... surface area airspace excluded below 1,100 feet MSL beyond 4 miles from the airport would be decreased... Designated as Surface Areas. * * * * * ] AAL AK E2 Kenai, AK Kenai Municipal Airport, AK (Lat. 60 34'24''...

  15. Comparison of actin and cell surface dynamics in motile fibroblasts

    PubMed Central

    1992-01-01

    We have investigated the dynamic behavior of actin in fibroblast lamellipodia using photoactivation of fluorescence. Activated regions of caged resorufin (CR)-labeled actin in lamellipodia of IMR 90 and MC7 3T3 fibroblasts were observed to move centripetally over time. Thus in these cells, actin filaments move centripetally relative to the substrate. Rates were characteristic for each cell type; 0.66 +/- 0.27 microns/min in IMR 90 and 0.36 +/- 0.16 microns/min in MC7 3T3 cells. In neither case was there any correlation between the rate of actin movement and the rate of lamellipodial protrusion. The half-life of the activated CR-actin filaments was approximately 1 min in IMR 90 lamellipodia, and approximately 3 min in MC7 3T3 lamellipodia. Thus continuous filament turnover accompanies centripetal movement. In both cell types, the length of time required for a section of the actin meshwork to traverse the lamellipodium was several times longer than the filament half-life. The dynamic behavior of the dorsal surface of the cell was also observed by tracking lectin-coated beads on the surface and phase-dense features within lamellipodia of MC7 3T3 cells. The movement of these dorsal features occurred at rates approximately three times faster than the rate of movement of the underlying bulk actin cytoskeleton, even when measured in the same individual cells. Thus the transport of these dorsal features must occur by some mechanism other than simple attachment to the moving bulk actin cytoskeleton. PMID:1400580

  16. Positioning and stretching of actin filaments by electric fields

    NASA Astrophysics Data System (ADS)

    Wigge, Christoph; Hinssen, Horst; Reiss, Günter; Herth, Simone

    2010-06-01

    The alignment of biological filaments on surfaces offers a high potential for controllable geometries in lab-on-a-chip-structures and micrototal analysis systems. Actin is a polar filamentous protein with a diameter of 7-8 nm that can be manipulated with strong electric fields. It is demonstrated that with the use of microelectrodes or nanoelectrodes and electric fields of 20 kV/m single actin filaments can be manipulated, stretched, and positioned between gold electrodes.

  17. Bulkiness or aromatic nature of tyrosine-143 of actin is important for the weak binding between F-actin and myosin-ADP-phosphate

    SciTech Connect

    Gomibuchi, Yuki; Uyeda, Taro Q.P.; Wakabayashi, Takeyuki

    2013-11-29

    Highlights: •The effect of mutation of Tyr143 that becomes more exposed on assembly was examined. •Mutation of tyrosine-143 of Dictyostelium actin changed actin polymerizability. •The bulkiness or aromatic nature of Tyr143 is important for the weak binding. •The weak interaction between myosin and actin strengthened by Tyr143Trp mutation. -- Abstract: Actin filaments (F-actin) interact with myosin and activate its ATPase to support force generation. By comparing crystal structures of G-actin and the quasi-atomic model of F-actin based on high-resolution cryo-electron microscopy, the tyrosine-143 was found to be exposed more than 60 Å{sup 2} to the solvent in F-actin. Because tyrosine-143 flanks the hydrophobic cleft near the hydrophobic helix that binds to myosin, the mutant actins, of which the tyrosine-143 was replaced with tryptophan, phenylalanine, or isoleucine, were generated using the Dictyostelium expression system. It polymerized significantly poorly when induced by NaCl, but almost normally by KCl. In the presence of phalloidin and KCl, the extents of the polymerization of all the mutant actins were comparable to that of the wild-type actin so that the actin-activated myosin ATPase activity could be reliably compared. The affinity of skeletal heavy meromyosin to F-actin and the maximum ATPase activity (V{sub max}) were estimated by a double reciprocal plot. The Tyr143Trp-actin showed the higher affinity (smaller K{sub app}) than that of the wild-type actin, with the V{sub max} being almost unchanged. The K{sub app} and V{sub max} of the Tyr143Phe-actin were similar to those of the wild-type actin. However, the activation by Tyr143Ile-actin was much smaller than the wild-type actin and the accurate determination of K{sub app} was difficult. Comparison of the myosin ATPase activated by the various mutant actins at the same concentration of F-actin showed that the extent of activation correlates well with the solvent-accessible surface areas (ASA

  18. In Vivo Imaging and Characterization of Actin Microridges

    PubMed Central

    Lam, Pui-ying; Mangos, Steve; Green, Julie M.; Reiser, Jochen; Huttenlocher, Anna

    2015-01-01

    Actin microridges form labyrinth like patterns on superficial epithelial cells across animal species. This highly organized assembly has been implicated in mucus retention and in the mechanical structure of mucosal surfaces, however the mechanisms that regulate actin microridges remain largely unknown. Here we characterize the composition and dynamics of actin microridges on the surface of zebrafish larvae using live imaging. Microridges contain phospho-tyrosine, cortactin and VASP, but not focal adhesion kinase. Time-lapse imaging reveals dynamic changes in the length and branching of microridges in intact animals. Transient perturbation of the microridge pattern occurs before cell division with rapid re-assembly during and after cytokinesis. Microridge assembly is maintained with constitutive activation of Rho or inhibition of myosin II activity. However, expression of dominant negative RhoA or Rac alters microridge organization, with an increase in distance between microridges. Latrunculin A treatment and photoconversion experiments suggest that the F-actin filaments are actively treadmilling in microridges. Accordingly, inhibition of Arp2/3 or PI3K signaling impairs microridge structure and length. Taken together, actin microridges in zebrafish represent a tractable in vivo model to probe pattern formation and dissect Arp2/3-mediated actin dynamics in vivo. PMID:25629723

  19. Regulation of Actin by Ion-Linked Equilibria

    PubMed Central

    Kang, Hyeran; Bradley, Michael J.; Elam, W. Austin; De La Cruz, Enrique M.

    2013-01-01

    Actin assembly, filament mechanical properties, and interactions with regulatory proteins depend on the types and concentrations of salts in solution. Salts modulate actin through both nonspecific electrostatic effects and specific binding to discrete sites. Multiple cation-binding site classes spanning a broad range of affinities (nanomolar to millimolar) have been identified on actin monomers and filaments. This review focuses on discrete, low-affinity cation-binding interactions that drive polymerization, regulate filament-bending mechanics, and modulate interactions with regulatory proteins. Cation binding may be perturbed by actin post-translational modifications and linked equilibria. Partial cation occupancy under physiological and commonly used in vitro solution conditions likely contribute to filament mechanical heterogeneity and structural polymorphism. Site-specific cation-binding residues are conserved in Arp2 and Arp3, and may play a role in Arp2/3 complex activation and actin-filament branching activity. Actin-salt interactions demonstrate the relevance of ion-linked equilibria in the operation and regulation of complex biological systems. PMID:24359734

  20. Actin Assembly at Model-Supported Lipid Bilayers

    PubMed Central

    Heath, George R.; Johnson, Benjamin R.G.; Olmsted, Peter D.; Connell, Simon D.; Evans, Stephen D.

    2013-01-01

    We report on the use of supported lipid bilayers to reveal dynamics of actin polymerization from a nonpolymerizing subphase via cationic phospholipids. Using varying fractions of charged lipid, lipid mobility, and buffer conditions, we show that dynamics at the nanoscale can be used to control the self-assembly of these structures. In the case of fluid-phase lipid bilayers, the actin adsorbs to form a uniform two-dimensional layer with complete surface coverage whereas gel-phase bilayers induce a network of randomly oriented actin filaments, of lower coverage. Reducing the pH increased the polymerization rate, the number of nucleation events, and the total coverage of actin. A model of the adsorption/diffusion process is developed to provide a description of the experimental data and shows that, in the case of fluid-phase bilayers, polymerization arises equally due to the adsorption and diffusion of surface-bound monomers and the addition of monomers directly from the solution phase. In contrast, in the case of gel-phase bilayers, polymerization is dominated by the addition of monomers from solution. In both cases, the filaments are stable for long times even when the G-actin is removed from the supernatant—making this a practical approach for creating stable lipid-actin systems via self-assembly. PMID:24268147

  1. Actin Is Involved in Auxin-Dependent Patterning1

    PubMed Central

    Maisch, Jan; Nick, Peter

    2007-01-01

    Polar transport of auxin has been identified as a central element of pattern formation. The polarity of auxin transport is linked to the cycling of pin-formed proteins, a process that is related to actomyosin-dependent vesicle traffic. To get insight into the role of actin for auxin transport, we used patterned cell division to monitor the polarity of auxin fluxes. We show that cell division in the tobacco (Nicotiana tabacum L. cv Bright-Yellow 2) cell line is partially synchronized and that this synchrony can be perturbed by inhibition of auxin transport by 1-N-naphthylphthalamic acid. To address the role of actin in this synchrony, we induced a bundled configuration of actin by overexpressing mouse talin. The bundling of actin impairs the synchrony of cell division and increases the sensitivity to 1-N-naphthylphthalamic acid. Addition of the polarly transported auxins indole-3-acetic acid and 1-naphthyl acetic acid (but not 2,4-dichlorophenoxyacetic acid) restored both the normal organization of actin and the synchrony of cell division. This study suggests that auxin controls its own transport by changing the state of actin filaments. PMID:17337532

  2. Actin is involved in auxin-dependent patterning.

    PubMed

    Maisch, Jan; Nick, Peter

    2007-04-01

    Polar transport of auxin has been identified as a central element of pattern formation. The polarity of auxin transport is linked to the cycling of pin-formed proteins, a process that is related to actomyosin-dependent vesicle traffic. To get insight into the role of actin for auxin transport, we used patterned cell division to monitor the polarity of auxin fluxes. We show that cell division in the tobacco (Nicotiana tabacum L. cv Bright-Yellow 2) cell line is partially synchronized and that this synchrony can be perturbed by inhibition of auxin transport by 1-N-naphthylphthalamic acid. To address the role of actin in this synchrony, we induced a bundled configuration of actin by overexpressing mouse talin. The bundling of actin impairs the synchrony of cell division and increases the sensitivity to 1-N-naphthylphthalamic acid. Addition of the polarly transported auxins indole-3-acetic acid and 1-naphthyl acetic acid (but not 2,4-dichlorophenoxyacetic acid) restored both the normal organization of actin and the synchrony of cell division. This study suggests that auxin controls its own transport by changing the state of actin filaments.

  3. Myelin basic protein domains involved in the interaction with actin.

    PubMed

    Roth, G A; Gonzalez, M D; Monferran, C G; De Santis, M L; Cumar, F A

    1993-11-01

    A fluorescence assay was used to measure the interaction of myelin basic protein (MBP) with monomeric actin labeled with a fluorescent compound (IAEDANS). The complex actin-IAEDANS increase the fluorescence in presence of MBP. The enhancement of the fluorescence has a sigmoidal dependence on the concentration of MBP and the fluorescence maximum is reached at a MBP:actin molar ratio of 1:20. The fluorescence maximum in absence of Ca2+ and ATP is 4 times lower than that in their presence although it is reached at the same MBP:actin molar ratio. Similar behavior is observed when synapsin replaces MBP, while acetylated MBP and bovine serum albumin fail to induce any fluorescence change. To define possible interacting domains on MBP involved in the actin-MBP interaction, experiments were performed using MBP-derived peptides obtained under controlled proteolysis of the whole molecule. The fluorescence changes induced by the different peptides depend on their location in the native protein and can not be explained simply by a difference in the net charge of the peptides. The results suggest that two sites are involved in the interaction. A Ca2+/ATP-dependent site located in the amino-terminal region (peptide 1-44) and a Ca2+/ATP-independent one near the carboxyl terminus of the MBP molecule. The actin-MBP interaction was also observed using immunoblot and ELISA techniques.

  4. Regulators of Actin Dynamics in Gastrointestinal Tract Tumors.

    PubMed

    Steinestel, Konrad; Wardelmann, Eva; Hartmann, Wolfgang; Grünewald, Inga

    2015-01-01

    Reorganization of the actin cytoskeleton underlies cell migration in a wide variety of physiological and pathological processes, such as embryonic development, wound healing, and tumor cell invasion. It has been shown that actin assembly and disassembly are precisely regulated by intracellular signaling cascades that respond to changes in the cell microenvironment, ligand binding to surface receptors, or oncogenic transformation of the cell. Actin-nucleating and actin-depolymerizing (ANFs/ADFs) and nucleation-promoting factors (NPFs) regulate cytoskeletal dynamics at the leading edge of migrating cells, thereby modulating cell shape; these proteins facilitate cellular movement and mediate degradation of the surrounding extracellular matrix by secretion of lytic proteases, thus eliminating barriers for tumor cell invasion. Accordingly, expression and activity of these actin-binding proteins have been linked to enhanced metastasis and poor prognosis in a variety of malignancies. In this review, we will summarize what is known about expression patterns and the functional role of actin regulators in gastrointestinal tumors and evaluate first pharmacological approaches to prevent invasion and metastatic dissemination of malignant cells. PMID:26345720

  5. Microstructure and Mechanical Properties of Composite Actin Networks

    NASA Astrophysics Data System (ADS)

    Gardel, Margaret; Shin, Jennifer; Mahadevan, L.; Matsudaira, Paul; Weitz, D. A.

    2003-03-01

    There exits a family of actin-binding proteins (ABPs) and each protein has a distinct function for bundling, networking, gelating, capping, or simply binding to actin. Whether actin serves as a structural or motile component, its mechanical properties are determined by its degree and kinds of association with different ABPs and these properties are often closely related to its functional needs. For instance, in a cell actin is highly crosslinked with multiple ABPs (fimbrin, alpha-actinin, etc.) to generate thrust and strength for locomotion. In the acrosomal reaction of horseshoe crab sperm, actin exists as a bundle of preassembled filaments crosslinked with scruin to form a rigid structure to penetrate into an egg without yielding. We study the effects three different ABPs (scruin,fimbrin and alpha-actinin) have on the rheology and microstructure of actin networks using multiparticle tracking, imaging, and bulk rheology. From these experiments we can deduce how an evolving microstructure affects the bulk rheological properties and the role different concentrations and kinds of ABPs have in these changes.

  6. Actin gene family dynamics in cryptomonads and red algae.

    PubMed

    Tanifuji, Goro; Archibald, John M

    2010-09-01

    Here we present evidence for a complex evolutionary history of actin genes in red algae and cryptomonads, a group that acquired photosynthesis secondarily through the engulfment of a red algal endosymbiont. Four actin genes were found in the nuclear genome of the cryptomonad, Guillardia theta, and in the genome of the red alga, Galdieria sulphuraria, a member of the Cyanidiophytina. Phylogenetic analyses reveal that the both organisms possess two distinct sequence types, designated "type-1" and "type-2." A weak but consistent phylogenetic affinity between the cryptomonad type-2 sequences and the type-2 sequences of G. sulphuraria and red algae belonging to the Rhodophytina was observed. This is consistent with the possibility that the cryptomonad type-2 sequences are derived from the red algal endosymbiont that gave rise to the cryptomonad nucleomorph and plastid. Red algae as a whole possess two very different actin sequence types, with G. sulphuraria being the only organism thus far known to possess both. The common ancestor of Rhodophytina and Cyanidiophytina may have had two actin genes, with differential loss explaining the distribution of these genes in modern-day groups. Our study provides new insight into the evolution and divergence of actin genes in cryptomonads and red algae, and in doing so underscores the challenges associated with heterogeneity in actin sequence evolution and ortholog/paralog detection.

  7. Single-Molecule Studies of Actin Assembly and Disassembly Factors

    PubMed Central

    Smith, Benjamin A.; Gelles, Jeff; Goode, Bruce L.

    2014-01-01

    The actin cytoskeleton is very dynamic and highly regulated by multiple associated proteins in vivo. Understanding how this system of proteins functions in the processes of actin network assembly and disassembly requires methods to dissect the mechanisms of activity of individual factors and of multiple factors acting in concert. The advent of single-filament and single-molecule fluorescence imaging methods has provided a powerful new approach to discovering actin-regulatory activities and obtaining direct, quantitative insights into the pathways of molecular interactions that regulate actin network architecture and dynamics. Here we describe techniques for acquisition and analysis of single-molecule data, applied to the novel challenges of studying the filament assembly and disassembly activities of actin-associated proteins in vitro. We discuss the advantages of single-molecule analysis in directly visualizing the order of molecular events, measuring the kinetic rates of filament binding and dissociation, and studying the coordination among multiple factors. The methods described here complement traditional biochemical approaches in elucidating actin-regulatory mechanisms in reconstituted filamentous networks. PMID:24630103

  8. Actin kinetics shapes cortical network structure and mechanics

    PubMed Central

    Fritzsche, Marco; Erlenkämper, Christoph; Moeendarbary, Emad; Charras, Guillaume; Kruse, Karsten

    2016-01-01

    The actin cortex of animal cells is the main determinant of cellular mechanics. The continuous turnover of cortical actin filaments enables cells to quickly respond to stimuli. Recent work has shown that most of the cortical actin is generated by only two actin nucleators, the Arp2/3 complex and the formin Diaph1. However, our understanding of their interplay, their kinetics, and the length distribution of the filaments that they nucleate within living cells is poor. Such knowledge is necessary for a thorough comprehension of cellular processes and cell mechanics from basic polymer physics principles. We determined cortical assembly rates in living cells by using single-molecule fluorescence imaging in combination with stochastic simulations. We find that formin-nucleated filaments are, on average, 10 times longer than Arp2/3-nucleated filaments. Although formin-generated filaments represent less than 10% of all actin filaments, mechanical measurements indicate that they are important determinants of cortical elasticity. Tuning the activity of actin nucleators to alter filament length distribution may thus be a mechanism allowing cells to adjust their macroscopic mechanical properties to their physiological needs. PMID:27152338

  9. Single Filaments to Reveal the Multiple Flavors of Actin.

    PubMed

    Jégou, Antoine; Romet-Lemonne, Guillaume

    2016-05-24

    A number of key cell processes rely on specific assemblies of actin filaments, which are all constructed from nearly identical building blocks: the abundant and extremely conserved actin protein. A central question in the field is to understand how different filament networks can coexist and be regulated. Discoveries in science are often related to technical advances. Here, we focus on the ongoing single filament revolution and discuss how these techniques have greatly contributed to our understanding of actin assembly. In particular, we highlight how they have refined our understanding of the many protein-based regulatory mechanisms that modulate actin assembly. It is now becoming apparent that other factors give filaments a specific identity that determines which proteins will bind to them. We argue that single filament techniques will play an essential role in the coming years as we try to understand the many ways actin filaments can take different flavors and unveil how these flavors modulate the action of regulatory proteins. We discuss different factors known to make actin filaments distinguishable by regulatory proteins and speculate on their possible consequences.

  10. Regulators of Actin Dynamics in Gastrointestinal Tract Tumors

    PubMed Central

    Steinestel, Konrad; Wardelmann, Eva; Hartmann, Wolfgang; Grünewald, Inga

    2015-01-01

    Reorganization of the actin cytoskeleton underlies cell migration in a wide variety of physiological and pathological processes, such as embryonic development, wound healing, and tumor cell invasion. It has been shown that actin assembly and disassembly are precisely regulated by intracellular signaling cascades that respond to changes in the cell microenvironment, ligand binding to surface receptors, or oncogenic transformation of the cell. Actin-nucleating and actin-depolymerizing (ANFs/ADFs) and nucleation-promoting factors (NPFs) regulate cytoskeletal dynamics at the leading edge of migrating cells, thereby modulating cell shape; these proteins facilitate cellular movement and mediate degradation of the surrounding extracellular matrix by secretion of lytic proteases, thus eliminating barriers for tumor cell invasion. Accordingly, expression and activity of these actin-binding proteins have been linked to enhanced metastasis and poor prognosis in a variety of malignancies. In this review, we will summarize what is known about expression patterns and the functional role of actin regulators in gastrointestinal tumors and evaluate first pharmacological approaches to prevent invasion and metastatic dissemination of malignant cells. PMID:26345720

  11. Cloning and characterization of an actin gene of Chlamys farreri and the phylogenetic analysis of mollusk actins

    NASA Astrophysics Data System (ADS)

    Ma, Hongming; Mai, Kangsen; Liufu, Zhiguo; Xu, Wei

    2007-07-01

    An actingene (CfACTI) was cloned by using RT-PCR, 3’ and 5’RACE from hemocytes of the sea scallop Chlamys farreri. The full length of the transcript is 1 535 bp, which contains a long 3’ un-translated region of 436bp and 59bp of a 5’ un-translated sequence. The open reading frame encodes a polypeptide of 376 amino acids. Sequence comparisons indicated that CfACTI is more closely related to vertebrate cytoplasmic actins than muscle types. Phylogenetic analysis showed that molluscan actins could be generally divided into two categories: muscle and cytoplasmic, although both are similar to vertebrate cytoplasmic actins. It was also inferred that different isotypes existed in muscle or cytoplasma in mollusks. The genomic sequence of CfACTI was cloned and sequenced. Only one intron was detected: it was located between codons 42 and 43 and different from vertebrate actin genes.

  12. Competition for actin between two distinct F-actin networks defines a bistable switch for cell polarization.

    PubMed

    Lomakin, Alexis J; Lee, Kun-Chun; Han, Sangyoon J; Bui, Duyen A; Davidson, Michael; Mogilner, Alex; Danuser, Gaudenz

    2015-11-01

    Symmetry-breaking polarization enables functional plasticity of cells and tissues and is yet not well understood. Here we show that epithelial cells, hard-wired to maintain a static morphology and to preserve tissue organization, can spontaneously switch to a migratory polarized phenotype after relaxation of the actomyosin cytoskeleton. We find that myosin II engages actin in the formation of cortical actomyosin bundles and thus makes it unavailable for deployment in the process of dendritic growth normally driving cell motility. Under low-contractility regimes, epithelial cells polarize in a front-back manner owing to the emergence of actin retrograde flows powered by dendritic polymerization of actin. Coupled to cell movement, the flows transport myosin II from the front to the back of the cell, where the motor locally 'locks' actin in contractile bundles. This polarization mechanism could be employed by embryonic and cancer epithelial cells in microenvironments where high-contractility-driven cell motion is inefficient.

  13. Dynamic Localization of G-actin During Membrane Protrusion in Neuronal Motility

    PubMed Central

    Lee, Chi Wai; Vitriol, Eric A.; Shim, Sangwoo; Wise, Ariel L.; Velayutham, Radhi P.; Zheng, James Q.

    2013-01-01

    Summary Background Actin-based cell motility is fundamental for the development, function, and malignant events of eukaryotic organisms. During neural development, axonal growth cones depend on rapid assembly and disassembly of actin filaments (F-actin) for their guided extension to specific targets for wiring. Monomeric globular actin (G-actin) is the building block for F-actin but is not considered to play a direct role in spatiotemporal control of actin dynamics in cell motility. Results Here we report that a pool of G-actin dynamically localizes to the leading edge of growth cones and neuroblastoma cells to spatially elevate the G-/F-actin ratio that drives membrane protrusion and cell movement. Loss of G-actin localization leads to the cessation and retraction of membrane protrusions. Moreover, G-actin localization occurs asymmetrically in growth cones during attractive turning. Finally, we identify the actin monomer binding proteins profilin and thymosin β4 as key molecules that localize actin monomers to the leading edge of lamellipodia for their motility. Conclusions Our results suggest that dynamic localization of G-actin provides a novel mechanism to regulate the spatiotemporal actin dynamics underlying membrane protrusion in cell locomotion and growth cone chemotaxis. PMID:23746641

  14. F-actin dismantling through a redox-driven synergy between Mical and cofilin.

    PubMed

    Grintsevich, Elena E; Yesilyurt, Hunkar Gizem; Rich, Shannon K; Hung, Ruei-Jiun; Terman, Jonathan R; Reisler, Emil

    2016-08-01

    Numerous cellular functions depend on actin filament (F-actin) disassembly. The best-characterized disassembly proteins, the ADF (actin-depolymerizing factor)/cofilins (encoded by the twinstar gene in Drosophila), sever filaments and recycle monomers to promote actin assembly. Cofilin is also a relatively weak actin disassembler, posing questions about mechanisms of cellular F-actin destabilization. Here we uncover a key link to targeted F-actin disassembly by finding that F-actin is efficiently dismantled through a post-translational-mediated synergism between cofilin and the actin-oxidizing enzyme Mical. We find that Mical-mediated oxidation of actin improves cofilin binding to filaments, where their combined effect dramatically accelerates F-actin disassembly compared with either effector alone. This synergism is also necessary and sufficient for F-actin disassembly in vivo, magnifying the effects of both Mical and cofilin on cellular remodelling, axon guidance and Semaphorin-Plexin repulsion. Mical and cofilin, therefore, form a redox-dependent synergistic pair that promotes F-actin instability by rapidly dismantling F-actin and generating post-translationally modified actin that has altered assembly properties. PMID:27454820

  15. Lifeact and Utr230 induce distinct actin assemblies in cell nuclei.

    PubMed

    Du, Jing; Fan, Yan-Lei; Chen, Tai-Lin; Feng, Xi-Qiao

    2015-11-01

    Nuclear actin assembly in somatic cells has been an enigma for a long time. Recently, with the advancement of novel F-actin probes, researchers have started to uncover this mystery. In this study, we investigated the actin dynamics in somatic cell nuclei using two probes: Lifeact and Utr230. Surprisingly, we observed that both Lifeact and Utr230 significantly interfered with actin dynamics in cell nuclei. Moreover, these two probes induced distinct patterns of nuclear actin assembly. While Lifeact induced filamentous actin assembly in cell nuclei, Utr230 led to various patterns of actin aggregates, including fibers, small puncta, and large patches. Moreover, the interference of actin dynamics by Lifeact was limited to nuclear actin, while Utr230 induced actin aggregation in both the nucleus and cytoplasm. Using time-lapse microscopy, we found that Lifeact-induced actin fibers remained steady over hours of observation, indicating a deficiency of nuclear F-actin reorganization. These results suggest that Lifeact and Utr230 both interfere with nuclear actin dynamics but with distinct mechanisms. This is an important finding for research on nuclear actin assembly and highlights the potential value of these two probes for exploring the native mechanisms underlying nuclear actin dynamics, which appear to be altered in the presence of these probes.

  16. Arabidopsis AtADF1 is functionally affected by mutations on actin binding sites.

    PubMed

    Dong, Chun-Hai; Tang, Wei-Ping; Liu, Jia-Yao

    2013-03-01

    The plant actin depolymerizing factor (ADF) binds to both monomeric and filamentous actin, and is directly involved in the depolymerization of actin filaments. To better understand the actin binding sites of the Arabidopsis thaliana L. AtADF1, we generated mutants of AtADF1 and investigated their functions in vitro and in vivo. Analysis of mutants harboring amino acid substitutions revealed that charged residues (Arg98 and Lys100) located at the α-helix 3 and forming an actin binding site together with the N-terminus are essential for both G- and F-actin binding. The basic residues on the β-strand 5 (K82/A) and the α-helix 4 (R135/A, R137/A) form another actin binding site that is important for F-actin binding. Using transient expression of CFP-tagged AtADF1 mutant proteins in onion (Allium cepa) peel epidermal cells and transgenic Arabidopsis thaliana L. plants overexpressing these mutants, we analyzed how these mutant proteins regulate actin organization and affect seedling growth. Our results show that the ADF mutants with a lower affinity for actin filament binding can still be functional, unless the affinity for actin monomers is also affected. The G-actin binding activity of the ADF plays an essential role in actin binding, depolymerization of actin polymers, and therefore in the control of actin organization. PMID:23190411

  17. Ha-VP39 binding to actin and the influence of F-actin on assembly of progeny virions.

    PubMed

    Lu, S; Ge, G; Qi, Y

    2004-11-01

    We present evidence that actin is necessary for the successful assembly of HaNPV virions. Purified nucleocapsid protein Ha-VP39 of Heliothis armigera nuclear polyhedrosis virus (HaNPV) was found to be able to bind to actin in vitro without assistance, as demonstrated by Western blot and isothermal titration calorimeter. DeltaH and binding constants (K) detected by isothermal titration calorimeter strongly suggested that Ha-VP39 first binds actin to seed the formation of hexamer complex of actin, and the hexamers then link to each other to form filaments, and the filaments finally twist into cable structures. The proliferation of HaNPV was completely inhibited in Hz-AM1 cells cultivated in the medium containing 0.5 microg/ml cytochalasin D (CD) to prevent polymerization of actin, while its yield was reduced to 10(-4) in the presence of 0.1 microg/ml CD. Actin concentration and the viral DNA synthesis were not significantly affected by CD even though the progeny virions assembled in the CD treated cells were morphologically different from normal ones and resulted in fewer plaques in plaque assay.

  18. Structure of the FMNL3 FH2/actin complex provides insight into formin-mediated actin nucleation and elongation

    PubMed Central

    Thompson, Morgan E.; Heimsath, Ernest G.; Gauvin, Timothy J.; Higgs, Henry N.; Kull, F. Jon

    2012-01-01

    Summary Formins are actin assembly factors that act in a variety of actin-based processes. The conserved formin homology 2 (FH2) domain promotes filament nucleation and influences elongation via interaction with the barbed end. FMNL3 is a formin that induces assembly of filopodia but whose FH2 domain is a poor nucleator. The 3.4 Å structure of an FMNL3 FH2 dimer in complex with tetramethylrhodamine-actin uncovers details of formin-regulated actin elongation. We observe distinct FH2-actin binding regions; interactions in the knob and coiled-coil subdomains are necessary for actin binding while those in the lasso/post interface are important for the stepping mechanism. Biochemical and cellular experiments test the importance of individual residues for function. This structure provides details for FH2 mediated filament elongation via processive capping and supports a model in which C-terminal non-FH2 residues of FMNL3 are required to stabilize the filament nucleus. PMID:23222643

  19. Multilayer defects nucleated by substrate pits: a comparison of actinic inspection and non-actinic inspection techniques

    SciTech Connect

    Barty, A; Goldberg, K; Kearney, P; Rekawa, S; LaFontaine, B; Wood, O; Taylor, J S; Han, H

    2006-10-02

    The production of defect-free mask blanks remains a key challenge for EUV lithography. Mask-blank inspection tools must be able to accurately detect all critical defects while simultaneously having the minimum possible false-positive detection rate. We have recently observed and here report the identification of bump-type buried substrate defects, that were below the detection limit of a non-actinic (i.e. non-EUV) in inspection tool. Presently, the occurrence inspection of pit-type defects, their printability, and their detectability with actinic techniques and non-actinic commercial tools, has become a significant concern. We believe that the most successful strategy for the development of effective non-actinic mask inspection tools will involve the careful cross-correlation with actinic inspection and lithographic printing. In this way, the true efficacy of prototype inspection tools now under development can be studied quantitatively against relevant benchmarks. To this end we have developed a dual-mode actinic mask inspection system capable of scanning mask blanks for defects (with simultaneous EUV bright-field and dark-field detection) and imaging those same defects with a zoneplate microscope that matches or exceeds the resolution of EUV steppers.

  20. Effect of ATP analogues on the actin-myosin interface.

    PubMed

    Van Dijk, J; Fernandez, C; Chaussepied, P

    1998-06-01

    The interaction between skeletal myosin subfragment 1 (S1) and filamentous actin was examined at various intermediate states of the actomyosin ATPase cycle by chemical cross-linking experiments. Reaction of the actin-S1 complex with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide and N-hydroxysuccinimide generated products with molecular masses of 165 and 175 kDa, in which S1 loops of residues 626-647 and 567-578 were cross-linked independently to the N-terminal segment of residues 1-12 of one actin monomer, and of 265 kDa, in which the two loops were bound to the N termini of two adjacent monomers. In strong-binding complexes, i.e., without nucleotide or with ADP, S1 was sequentially cross-linked to one and then to two actin monomers. In the weak-binding complexes, two types of cross-linking pattern were observed. First, during steady-state hydrolysis of ATP or ATPgammaS at 20 degreesC, the cross-linking reaction gave rise to a small amount of unknown 200 kDa product. Second, in the presence of AMPPNP, ADP.BeFx, ADP.AlF4-, or ADP.VO43- or with S1 internally cross-linked by N,N'-p-phenylenedimaleimide, only the 265 kDa product was obtained. The presence of 200 mM salt inhibited cross-linking reactions in both weak- and strong-binding states, while it dissociated only weak-binding complexes. These results indicate that, in the weak-binding state populated with the ADP.Pi analogues, skeletal S1 interacts predominantly and with an apparent equal affinity with the N termini of two adjacent actin monomers, while these ionic contacts are much less significant in stabilizing the rigor actin-S1 complexes. They also suggest that the electrostatic actin-S1 interface is not influenced by the type of ADP.Pi analogue bound to the active site.

  1. Modulation of the interaction between G-actin and thymosin beta 4 by the ATP/ADP ratio: possible implication in the regulation of actin dynamics.

    PubMed Central

    Carlier, M F; Jean, C; Rieger, K J; Lenfant, M; Pantaloni, D

    1993-01-01

    The interaction of G-actin with thymosin beta 4 (T beta 4), the major G-actin-sequestering protein in motile and proliferating cells, has been analyzed in vitro. T beta 4 is found to have a 50-fold higher affinity for MgATP-actin than for MgADP-actin. These results imply that in resting platelets and neutrophils, actin is sequestered by T beta 4 as MgATP-G-actin. Kinetic experiments and theoretical calculations demonstrate that this ATP/ADP dependence of T beta 4 affinity for G-actin can generate a mechanism of desequestration of G-actin by ADP, in the presence of physiological concentrations of T beta 4 (approximately 0.1 mM). The desequestration of G-actin by ADP is kinetically enhanced by profilin, which accelerates the dissociation of ATP from G-actin. Whether a local drop in the ATP/ADP ratio can allow local, transient desequestration and polymerization of actin either close to the plasma membrane, following platelet or neutrophil stimulation, or behind the Listeria bacterium in the host cell, while the surrounding cytoplasm contains sequestered ATP-G-actin, is an open issue raised by the present work. PMID:8506348

  2. A central role for the WH2 domain of Srv2/CAP in recharging actin monomers to drive actin turnover in vitro and in vivo

    PubMed Central

    Chaudhry, Faisal; Little, Kristin; Talarico, Lou; Quintero-Monzon, Omar; Goode, Bruce L.

    2010-01-01

    Cellular processes propelled by actin polymerization require rapid disassembly of filaments, and then efficient recycling of ADF/cofilin-bound ADP-actin monomers back to an assembly-competent ATP-bound state. How monomer recharging is regulated in vivo is still not well understood, but recent work suggests the involvement of the ubiquitous actin-monomer binding protein Srv2/CAP. To better understand Srv2/CAP mechanism, we explored the contribution of its WH2 domain, the function of which has remained highly elusive. We found that the WH2 domain binds to actin monomers and, unlike most other WH2 domains, exhibits similar binding affinity for ATP-actin and ADP-actin (Kd ~1.5μM). Mutations in the WH2 domain that impair actin binding disrupt the ability of purified full-length Srv2/CAP to catalyze nucleotide exchange on ADF/cofilin-bound actin monomers and accelerate actin turnover in vitro. The same mutations impair Srv2/CAP function in vivo in regulating actin organization, cell growth, and cell morphogenesis. Thus, normal cell growth and organization depend on the ability of Srv2/CAP to recharge actin monomers, and the WH2 domain plays a central role in this process. Our data also reveal that while most isolated WH2 domains inhibit nucleotide exchange on actin, WH2 domains in the context of intact proteins can help promote nucleotide exchange. PMID:20169536

  3. Structure of a pentavalent G-actin*MRTF-A complex reveals how G-actin controls nucleocytoplasmic shuttling of a transcriptional coactivator.

    PubMed

    Mouilleron, Stéphane; Langer, Carola A; Guettler, Sebastian; McDonald, Neil Q; Treisman, Richard

    2011-06-14

    Subcellular localization of the actin-binding transcriptional coactivator MRTF-A is controlled by its interaction with monomeric actin (G-actin). Signal-induced decreases in G-actin concentration reduce MRTF-A nuclear export, leading to its nuclear accumulation, whereas artificial increases in G-actin concentration in resting cells block MRTF-A nuclear import, retaining it in the cytoplasm. This regulation is dependent on three actin-binding RPEL motifs in the regulatory domain of MRTF-A. We describe the structures of pentavalent and trivalent G-actin•RPEL domain complexes. In the pentavalent complex, each RPEL motif and the two intervening spacer sequences bound an actin monomer, forming a compact assembly. In contrast, the trivalent complex lacked the C-terminal spacer- and RPEL-actins, both of which bound only weakly in the pentavalent complex. Cytoplasmic localization of MRTF-A in unstimulated fibroblasts also required binding of G-actin to the spacer sequences. The bipartite MRTF-A nuclear localization sequence was buried in the pentameric assembly, explaining how increases in G-actin concentration prevent nuclear import of MRTF-A. Analyses of the pentavalent and trivalent complexes show how actin loads onto the RPEL domain and reveal a molecular mechanism by which actin can control the activity of one of its binding partners.

  4. Incorporation and turnover of biotin-labeled actin microinjected into fibroblastic cells: an immunoelectron microscopic study

    PubMed Central

    1989-01-01

    We investigated the mechanism of turnover of an actin microfilament system in fibroblastic cells on an electron microscopic level. A new derivative of actin was prepared by labeling muscle actin with biotin. Cultured fibroblastic cells were microinjected with biotinylated actin, and incorporated biotin-actin molecules were detected by immunoelectron microscopy using an anti-biotin antibody and a colloidal gold-labeled secondary antibody. We also analyzed the localization of injected biotin-actin molecules on a molecular level by freeze-drying techniques. Incorporation of biotin-actin was rapid in motile peripheral regions, such as lamellipodia and microspikes. At approximately 1 min after injection, biotin-actin molecules were mainly incorporated into the distal part of actin bundles in the microspikes. Heavily labeled actin filaments were also observed at the distal fringe of the densely packed actin networks in the lamellipodium. By 5 min after injection, most actin polymers in microspikes and lamellipodia were labeled uniformly. These findings suggest that actin subunits are added preferentially at the membrane-associated ends of preexisting actin filaments. At earlier times after injection, we often observed that the labeled segments were continuous with unlabeled segments, suggesting the incorporation of new subunits at the ends of preexisting filaments. Actin incorporation into stress fibers was a slower process. At 2-3 min after injection, microfilaments at the surface of stress fibers incorporated biotin-actin, but filaments in the core region of stress fibers did not. At 5-10 min after injection, increasing density of labeling along stress fibers toward their distal ends was observed. Stress fiber termini are generally associated with focal contacts. There was no rapid nucleation of actin filaments off the membrane of focal contacts and the pattern of actin incorporation at focal contacts was essentially identical to that into distal parts of stress fibers

  5. How actin binds and assembles onto plasma membranes from Dictyostelium discoideum

    PubMed Central

    1988-01-01

    We have shown previously (Schwartz, M. A., and E. J. Luna. 1986. J. Cell Biol. 102: 2067-2075) that actin binds with positive cooperativity to plasma membranes from Dictyostelium discoideum. Actin is polymerized at the membrane surface even at concentrations well below the critical concentration for polymerization in solution. Low salt buffer that blocks actin polymerization in solution also prevents actin binding to membranes. To further explore the relationship between actin polymerization and binding to membranes, we prepared four chemically modified actins that appear to be incapable of polymerizing in solution. Three of these derivatives also lost their ability to bind to membranes. The fourth derivative (EF actin), in which histidine-40 is labeled with ethoxyformic anhydride, binds to membranes with reduced affinity. Binding curves exhibit positive cooperativity, and cross- linking experiments show that membrane-bound actin is multimeric. Thus, binding and polymerization are tightly coupled, and the ability of these membranes to polymerize actin is dramatically demonstrated. EF actin coassembles weakly with untreated actin in solution, but coassembles well on membranes. Binding by untreated actin and EF actin are mutually competitive, indicating that they bind to the same membrane sites. Hill plots indicate that an actin trimer is the minimum assembly state required for tight binding to membranes. The best explanation for our data is a model in which actin oligomers assemble by binding to clustered membrane sites with successive monomers on one side of the actin filament bound to the membrane. Individual binding affinities are expected to be low, but the overall actin-membrane avidity is high, due to multivalency. Our results imply that extracellular factors that cluster membrane proteins may create sites for the formation of actin nuclei and thus trigger actin polymerization in the cell. PMID:3392099

  6. Modulation of actin structure and function by phosphorylation of Tyr-53 and profilin binding

    SciTech Connect

    Baek, Kyuwon; Liu, Xiong; Ferron, Francois; Shu, Shi; Korn, Edward D.; Dominguez, Roberto

    2008-08-27

    On starvation, Dictyostelium cells aggregate to form multicellular fruiting bodies containing spores that germinate when transferred to nutrient-rich medium. This developmental cycle correlates with the extent of actin phosphorylation at Tyr-53 (pY53-actin), which is low in vegetative cells but high in viable mature spores. Here we describe high-resolution crystal structures of pY53-actin and unphosphorylated actin in complexes with gelsolin segment 1 and profilin. In the structure of pY53-actin, the phosphate group on Tyr-53 makes hydrogen-bonding interactions with residues of the DNase I-binding loop (D-loop) of actin, resulting in a more stable conformation of the D-loop than in the unphosphorylated structures. A more rigidly folded D-loop may explain some of the previously described properties of pY53-actin, including its increased critical concentration for polymerization, reduced rates of nucleation and pointed end elongation, and weak affinity for DNase I. We show here that phosphorylation of Tyr-53 inhibits subtilisin cleavage of the D-loop and reduces the rate of nucleotide exchange on actin. The structure of profilin-Dictyostelium-actin is strikingly similar to previously determined structures of profilin-{beta}-actin and profilin-{alpha}-actin. By comparing this representative set of profilin-actin structures with other structures of actin, we highlight the effects of profilin on the actin conformation. In the profilin-actin complexes, subdomains 1 and 3 of actin close around profilin, producing a 4.7 deg. rotation of the two major domains of actin relative to each other. As a result, the nucleotide cleft becomes moderately more open in the profilin-actin complex, probably explaining the stimulation of nucleotide exchange on actin by profilin.

  7. Enhanced gravitropism of roots with a disrupted cap actin cytoskeleton

    NASA Technical Reports Server (NTRS)

    Hou, Guichuan; Mohamalawari, Deepti R.; Blancaflor, Elison B.

    2003-01-01

    The actin cytoskeleton has been proposed to be a major player in plant gravitropism. However, understanding the role of actin in this process is far from complete. To address this problem, we conducted an analysis of the effect of Latrunculin B (Lat B), a potent actin-disrupting drug, on root gravitropism using various parameters that included detailed curvature kinetics, estimation of gravitropic sensitivity, and monitoring of curvature development after extended clinorotation. Lat B treatment resulted in a promotion of root curvature after a 90 degrees reorientation in three plant species tested. More significantly, the sensitivity of maize (Zea mays) roots to gravity was enhanced after actin disruption, as determined from a comparison of presentation time of Lat B-treated versus untreated roots. A short 10-min gravistimulus followed by extended rotation on a 1-rpm clinostat resulted in extensive gravitropic responses, manifested as curvature that often exceeded 90 degrees. Application of Lat B to the cap or elongation zone of maize roots resulted in the disruption of the actin cytoskeleton, which was confined to the area of localized Lat B application. Only roots with Lat B applied to the cap displayed the strong curvature responses after extended clinorotation. Our study demonstrates that disrupting the actin cytoskeleton in the cap leads to the persistence of a signal established by a previous gravistimulus. Therefore, actin could function in root gravitropism by providing a mechanism to regulate the proliferation of a gravitropic signal originating from the cap to allow the root to attain its correct orientation or set point angle.

  8. Enhanced gravitropism of roots with a disrupted cap actin cytoskeleton.

    PubMed

    Hou, Guichuan; Mohamalawari, Deepti R; Blancaflor, Elison B

    2003-03-01

    The actin cytoskeleton has been proposed to be a major player in plant gravitropism. However, understanding the role of actin in this process is far from complete. To address this problem, we conducted an analysis of the effect of Latrunculin B (Lat B), a potent actin-disrupting drug, on root gravitropism using various parameters that included detailed curvature kinetics, estimation of gravitropic sensitivity, and monitoring of curvature development after extended clinorotation. Lat B treatment resulted in a promotion of root curvature after a 90 degrees reorientation in three plant species tested. More significantly, the sensitivity of maize (Zea mays) roots to gravity was enhanced after actin disruption, as determined from a comparison of presentation time of Lat B-treated versus untreated roots. A short 10-min gravistimulus followed by extended rotation on a 1-rpm clinostat resulted in extensive gravitropic responses, manifested as curvature that often exceeded 90 degrees. Application of Lat B to the cap or elongation zone of maize roots resulted in the disruption of the actin cytoskeleton, which was confined to the area of localized Lat B application. Only roots with Lat B applied to the cap displayed the strong curvature responses after extended clinorotation. Our study demonstrates that disrupting the actin cytoskeleton in the cap leads to the persistence of a signal established by a previous gravistimulus. Therefore, actin could function in root gravitropism by providing a mechanism to regulate the proliferation of a gravitropic signal originating from the cap to allow the root to attain its correct orientation or set point angle. PMID:12644685

  9. Actin polymerization and intracellular solvent flow in cell surface blebbing

    PubMed Central

    1995-01-01

    The cortical actin gel of eukaryotic cells is postulated to control cell surface activity. One type of protrusion that may offer clues to this regulation are the spherical aneurysms of the surface membrane known as blebs. Blebs occur normally in cells during spreading and alternate with other protrusions, such as ruffles, suggesting similar protrusive machinery is involved. We recently reported that human melanoma cell lines deficient in the actin filament cross-linking protein, ABP-280, show prolonged blebbing, thus allowing close study of blebs and their dynamics. Blebs expand at different rates of volume increase that directly predict the final size achieved by each bleb. These rates decrease as the F-actin concentration of the cells increase over time after plating on a surface, but do so at lower concentrations in ABP-280 expressing cells. Fluorescently labeled actin and phalloidin injections of blebbing cells indicate that a polymerized actin structure is not present initially, but appears later and is responsible for stopping further bleb expansion. Therefore, it is postulated that blebs occur when the fluid-driven expansion of the cell membrane is sufficiently rapid to initially outpace the local rate of actin polymerization. In this model, the rate of intracellular solvent flow driving this expansion decreases as cortical gelation is achieved, whether by factors such as ABP-280, or by concentrated actin polymers alone, thereby leading to decreased size and occurrence of blebs. Since the forces driving bleb extension would always be present in a cell, this process may influence other cell protrusions as well. PMID:7790356

  10. Mechanics of Biomimetic Liposomes Encapsulating an Actin Shell.

    PubMed

    Guevorkian, Karine; Manzi, John; Pontani, Léa-Lætitia; Brochard-Wyart, Françoise; Sykes, Cécile

    2015-12-15

    Cell-shape changes are insured by a thin, dynamic, cortical layer of cytoskeleton underneath the plasma membrane. How this thin cortical structure impacts the mechanical properties of the whole cell is not fully understood. Here, we study the mechanics of liposomes or giant unilamellar vesicles, when a biomimetic actin cortex is grown at the inner layer of the lipid membrane via actin-nucleation-promoting factors. Using a hydrodynamic tube-pulling technique, we show that tube dynamics is clearly affected by the presence of an actin shell anchored to the lipid bilayer. The same force pulls much shorter tubes in the presence of the actin shell compared to bare membranes. However, in both cases, we observe that the dynamics of tube extrusion has two distinct features characteristic of viscoelastic materials: rapid elastic elongation, followed by a slower elongation phase at a constant rate. We interpret the initial elastic regime by an increase of membrane tension due to the loss of lipids into the tube. Tube length is considerably shorter for cortex liposomes at comparable pulling forces, resulting in a higher spring constant. The presence of the actin shell seems to restrict lipid mobility, as is observed in the corral effect in cells. The viscous regime for bare liposomes corresponds to a leakout of the internal liquid at constant membrane tension. The presence of the actin shell leads to a larger friction coefficient. As the tube is pulled from a patchy surface, membrane tension increases locally, leading to a Marangoni flow of lipids. As a conclusion, the presence of an actin shell is revealed by its action that alters membrane mechanics.

  11. Actin-cytoskeleton rearrangement modulates proton-induced uptake

    SciTech Connect

    Ben-Dov, Nadav; Korenstein, Rafi

    2013-04-15

    Recently it has been shown that elevating proton concentration at the cell surface stimulates the formation of membrane invaginations and vesicles accompanied by an enhanced uptake of macromolecules. While the initial induction of inward membrane curvature was rationalized in terms of proton-based increase of charge asymmetry across the membrane, the mechanisms underlying vesicle formation and its scission are still unknown. In light of the critical role of actin in vesicle formation during endocytosis, the present study addresses the involvement of cytoskeletal actin in proton-induced uptake (PIU). The uptake of dextran-FITC is used as a measure for the factual fraction of inward invaginations that undergo scission from the cell's plasma membrane. Our findings show that the rate of PIU in suspended cells is constant, whereas the rate of PIU in adherent cells is gradually increased in time, saturating at the level possessed by suspended cells. This is consistent with pH induced gradual degradation of stress-fibers in adherent cells. Wortmannin and calyculin-A are able to elevate PIU by 25% in adherent cells but not in suspended cells, while cytochalasin-D, rapamycin and latrunculin-A elevate PIU both in adherent and suspended cells. However, extensive actin depolymerization by high concentrations of latrunculin-A is able to inhibit PIU. We conclude that proton-induced membrane vesiculation is restricted by the actin structural resistance to the plasma membrane bending. Nevertheless, a certain degree of cortical actin restructuring is required for the completion of the scission process. - Highlights: ► Acidification of cells' exterior enhances uptake of macromolecules by the cells. ► Disruption of actin stress fibers leads to enhancement of proton induced uptake. ► Extensive depolymerization of cellular actin attenuates proton-induced uptake.

  12. Optogenetics to target actin-mediated synaptic loss in Alzheimer's

    NASA Astrophysics Data System (ADS)

    Zahedi, Atena; DeFea, Kathryn; Ethell, Iryna

    2013-03-01

    Numerous studies in Alzheimer's Disease (AD) animal models show that overproduction of Aβ peptides and their oligomerization can distort dendrites, damage synapses, and decrease the number of dendritic spines and synapses. Aβ may trigger synapse loss by modulating activity of actin-regulating proteins, such as Rac1 and cofilin. Indeed, Aβ1-42 oligomers can activate actin severing protein cofilin through calcineurin-mediated activation of phosphatase slingshot and inhibit an opposing pathway that suppresses cofilin phosphorylation through Rac-mediated activation of LIMK1. Excessive activation of actin-severing protein cofilin triggers the formation of a non-dynamic actin bundles, called rods that are found in AD brains and cause loss of synapses. Hence, regulation of these actin-regulating proteins in dendritic spines could potentially provide useful tools for preventing the synapse/spine loss associated with earlier stages of AD neuropathology. However, lack of spatiotemporal control over their activity is a key limitation. Recently, optogenetic advancements have provided researchers with convenient light-activating proteins such as photoactivatable Rac (PARac). Here, we transfected cultured primary hippocampal neurons and human embryonic kidney (HEK) cells with a PARac/ mCherry-containing plasmid and the mCherry-positive cells were identified and imaged using an inverted fluorescence microscope. Rac1 activation was achieved by irradiation with blue light (480nm) and live changes in dendritic spine morphology were observed using mCherry (587nm). Rac activation was confirmed by immunostaining for phosphorylated form of effector proteinP21 protein-activated kinase 1 (PAK1) and reorganization of actin. Thus, our studies confirm the feasibility of using the PA-Rac construct to trigger actin re-organization in the dendritic spines.

  13. Clamped-filament elongation model for actin-based motors.

    PubMed Central

    Dickinson, Richard B; Purich, Daniel L

    2002-01-01

    Although actin-based motility drives cell crawling and intracellular locomotion of organelles and certain pathogens, the underlying mechanism of force generation remains a mystery. Recent experiments demonstrated that Listeria exhibit episodes of 5.4-nm stepwise motion corresponding to the periodicity of the actin filament subunits, and extremely small positional fluctuations during the intermittent pauses [S. C. Kuo and J. L. McGrath. 2000. Nature. 407:1026-1029]. These findings suggest that motile bacteria remain firmly bound to actin filament ends as they elongate, a behavior that appears to rule out previous models for actin-based motility. We propose and analyze a new mechanochemical model (called the "Lock, Load & Fire" mechanism) for force generation by means of affinity-modulated, clamped-filament elongation. During the locking step, the filament's terminal ATP-containing subunit binds tightly to a clamp situated on the surface of a motile object; in the loading step, actin.ATP monomer(s) bind to the filament end, an event that triggers the firing step, wherein ATP hydrolysis on the clamped subunit attenuates the filament's affinity for the clamp. This last step initiates translocation of the new ATP-containing terminus to the clamp, whereupon another cycle begins anew. This model explains how surface-tethered filaments can grow while exerting flexural or tensile force on the motile surface. Moreover, stochastic simulations of the model reproduce the signature motions of Listeria. This elongation motor, which we term actoclampin, exploits actin's intrinsic ATPase activity to provide a simple, high-fidelity enzymatic reaction cycle for force production that does not require elongating filaments to dissociate from the motile surface. This mechanism may operate whenever actin polymerization is called upon to generate the forces that drive cell crawling or intracellular organelle motility. PMID:11806905

  14. Actin expression in germinating seeds of Phaseolus vulgaris L.

    PubMed

    Villanueva, M A; Campos, F; Díaz, C; Colmenero-Flores, J M; Dantán, E; Sánchez, F; Covarrubias, A A

    1999-02-01

    Actin was present at very low levels in the seeds of common bean (Phaseolus vulgaris L.) compared with those from other species, and was observed mostly in the embryo. A time-course of actin expression in germinating bean seeds revealed an induced expression of both the mRNA and protein. Initially, the actin mRNA in seeds was barely detectable by northern blot analysis. However, there was a substantial increase in the expression of the actin mRNA at 24, 48 and 72 h after imbibition, compared with an internal control consisting of a late-embryogenesis-abundant (LEA) type IV gene from P. vulgaris. An increase in the amount of actin in total seed extracts that parallelled that of the mRNA was detected by western blotting starting at 24 h after imbibition. This increase was more apparent when the embryo alone was analyzed. Two-dimensional west-ern blots initially revealed three actin isoforms with isoelectric points (pIs) of approximately 5.6, 5.7 and 5.8,the amounts of which increased within a 48-h period,when a new minor isoform of pI approximately 5.5 appeared; however, after 72 h, the pI-5.8 isoform had almost disappeared and the pI-5.5 isoform had disappeared completely, indicating that these two minor isoforms are expressed transiently. These results indicate that actin is at very low levels in the dry seed but undergoes an increased and differential expression during imbibition, an event probably required to carry out all the necessary functions for germination.

  15. The Nucleocapsid Domain of Gag Is Dispensable for Actin Incorporation into HIV-1 and for Association of Viral Budding Sites with Cortical F-Actin

    PubMed Central

    Stauffer, Sarah; Rahman, Sheikh Abdul; de Marco, Alex; Carlson, Lars-Anders; Glass, Bärbel; Oberwinkler, Heike; Herold, Nikolas; Briggs, John A. G.; Müller, Barbara

    2014-01-01

    ABSTRACT Actin and actin-binding proteins are incorporated into HIV-1 particles, and F-actin has been suggested to bind the NC domain in HIV-1 Gag. Furthermore, F-actin has been frequently observed in the vicinity of HIV-1 budding sites by cryo-electron tomography (cET). Filamentous structures emanating from viral buds and suggested to correspond to actin filaments have been observed by atomic force microscopy. To determine whether the NC domain of Gag is required for actin association with viral buds and for actin incorporation into HIV-1, we performed comparative analyses of virus-like particles (VLPs) obtained by expression of wild-type HIV-1 Gag or a Gag variant where the entire NC domain had been replaced by a dimerizing leucine zipper [Gag(LZ)]. The latter protein yielded efficient production of VLPs with near-wild-type assembly kinetics and size and exhibited a regular immature Gag lattice. Typical HIV-1 budding sites were detected by using cET in cells expressing either Gag or Gag(LZ), and no difference was observed regarding the association of buds with the F-actin network. Furthermore, actin was equally incorporated into wild-type HIV-1 and Gag- or Gag(LZ)-derived VLPs, with less actin per particle observed than had been reported previously. Incorporation appeared to correlate with the relative intracellular actin concentration, suggesting an uptake of cytosol rather than a specific recruitment of actin. Thus, the NC domain in HIV-1 Gag does not appear to have a role in actin recruitment or actin incorporation into HIV-1 particles. IMPORTANCE HIV-1 particles bud from the plasma membrane, which is lined by a network of actin filaments. Actin was found to interact with the nucleocapsid domain of the viral structural protein Gag and is incorporated in significant amounts into HIV-1 particles, suggesting that it may play an active role in virus release. Using electron microscopy techniques, we previously observed bundles of actin filaments near HIV-1 buds

  16. The Effects of Disease Models of Nuclear Actin Polymerization on the Nucleus

    PubMed Central

    Serebryannyy, Leonid A.; Yuen, Michaela; Parilla, Megan; Cooper, Sandra T.; de Lanerolle, Primal

    2016-01-01

    Actin plays a crucial role in regulating multiple processes within the nucleus, including transcription and chromatin organization. However, the polymerization state of nuclear actin remains controversial, and there is no evidence for persistent actin filaments in a normal interphase nucleus. Further, several disease pathologies are characterized by polymerization of nuclear actin into stable filaments or rods. These include filaments that stain with phalloidin, resulting from point mutations in skeletal α-actin, detected in the human skeletal disease intranuclear rod myopathy, and cofilin/actin rods that form in response to cellular stressors like heatshock. To further elucidate the effects of these pathological actin structures, we examined the nucleus in both cell culture models as well as isolated human tissues. We find these actin structures alter the distribution of both RNA polymerase II and chromatin. Our data suggest that nuclear actin filaments result in disruption of nuclear organization, which may contribute to the disease pathology. PMID:27774069

  17. Bundling of actin filaments by elongation factor 1 alpha inhibits polymerization at filament ends

    PubMed Central

    1996-01-01

    Elongation factor 1 alpha (EF1 alpha) is an abundant protein that binds aminoacyl-tRNA and ribosomes in a GTP-dependent manner. EF1 alpha also interacts with the cytoskeleton by binding and bundling actin filaments and microtubules. In this report, the effect of purified EF1 alpha on actin polymerization and depolymerization is examined. At molar ratios present in the cytosol, EF1 alpha significantly blocks both polymerization and depolymerization of actin filaments and increases the final extent of actin polymer, while at high molar ratios to actin, EF1 alpha nucleates actin polymerization. Although EF1 alpha binds actin monomer, this monomer-binding activity does not explain the effects of EF1 alpha on actin polymerization at physiological molar ratios. The mechanism for the inhibition of polymerization is related to the actin-bundling activity of EF1 alpha. Both ends of the actin filament are inhibited for polymerization and both bundling and the inhibition of actin polymerization are affected by pH within the same physiological range; at high pH both bundling and the inhibition of actin polymerization are reduced. Additionally, it is seen that the binding of aminoacyl-tRNA to EF1 alpha releases EF1 alpha's inhibiting effect on actin polymerization. These data demonstrate that EF1 alpha can alter the assembly of F-actin, a filamentous scaffold on which non- membrane-associated protein translation may be occurring in vivo. PMID:8947553

  18. Oscillatory increases in alkalinity anticipate growth and may regulate actin dynamics in pollen tubes of lily.

    PubMed

    Lovy-Wheeler, Alenka; Kunkel, Joseph G; Allwood, Ellen G; Hussey, Patrick J; Hepler, Peter K

    2006-09-01

    Lily (Lilium formosanum or Lilium longiflorum) pollen tubes, microinjected with a low concentration of the pH-sensitive dye bis-carboxyethyl carboxyfluorescein dextran, show oscillating pH changes in their apical domain relative to growth. An increase in pH in the apex precedes the fastest growth velocities, whereas a decline follows growth, suggesting a possible relationship between alkalinity and cell extension. A target for pH may be the actin cytoskeleton, because the apical cortical actin fringe resides in the same region as the alkaline band in lily pollen tubes and elongation requires actin polymerization. A pH-sensitive actin binding protein, actin-depolymerizing factor (ADF), together with actin-interacting protein (AIP) localize to the cortical actin fringe region. Modifying intracellular pH leads to reorganization of the actin cytoskeleton, especially in the apical domain. Acidification causes actin filament destabilization and inhibits growth by 80%. Upon complete growth inhibition, the actin fringe is the first actin cytoskeleton component to disappear. We propose that during normal growth, the pH increase in the alkaline band stimulates the fragmenting activity of ADF/AIP, which in turn generates more sites for actin polymerization. Increased actin polymerization supports faster growth rates and a proton influx, which inactivates ADF/AIP, decreases actin polymerization, and retards growth. As pH stabilizes and increases, the activity of ADF/AIP again increases, repeating the cycle of events. PMID:16920777

  19. Actin filament organization in the fish keratocyte lamellipodium

    PubMed Central

    1995-01-01

    From recent studies of locomoting fish keratocytes it was proposed that the dynamic turnover of actin filaments takes place by a nucleation- release mechanism, which predicts the existence of short (less than 0.5 microns) filaments throughout the lamellipodium (Theriot, J. A., and T. J. Mitchison. 1991. Nature (Lond.). 352:126-131). We have tested this model by investigating the structure of whole mount keratocyte cytoskeletons in the electron microscope and phalloidin-labeled cells, after various fixations, in the light microscope. Micrographs of negatively stained keratocyte cytoskeletons produced by Triton extraction showed that the actin filaments of the lamellipodium are organized to a first approximation in a two-dimensional orthogonal network with the filaments subtending an angle of around 45 degrees to the cell front. Actin filament fringes grown onto the front edge of keratocyte cytoskeletons by the addition of exogenous actin showed a uniform polarity when decorated with myosin subfragment-1, consistent with the fast growing ends of the actin filaments abutting the anterior edge. A steady drop in filament density was observed from the mid- region of the lamellipodium to the perinuclear zone and in images of the more posterior regions of lower filament density many of the actin filaments could be seen to be at least several microns in length. Quantitative analysis of the intensity distribution of fluorescent phalloidin staining across the lamellipodium revealed that the gradient of filament density as well as the absolute content of F-actin was dependent on the fixation method. In cells first fixed and then extracted with Triton, a steep gradient of phalloidin staining was observed from the front to the rear of the lamellipodium. With the protocol required to obtain the electron microscope images, namely Triton extraction followed by fixation, phalloidin staining was, significantly and preferentially reduced in the anterior part of the lamellipodium. This

  20. Cortactin promotes exosome secretion by controlling branched actin dynamics.

    PubMed

    Sinha, Seema; Hoshino, Daisuke; Hong, Nan Hyung; Kirkbride, Kellye C; Grega-Larson, Nathan E; Seiki, Motoharu; Tyska, Matthew J; Weaver, Alissa M

    2016-07-18

    Exosomes are extracellular vesicles that influence cellular behavior and enhance cancer aggressiveness by carrying bioactive molecules. The mechanisms that regulate exosome secretion are poorly understood. Here, we show that the actin cytoskeletal regulatory protein cortactin promotes exosome secretion. Knockdown or overexpression of cortactin in cancer cells leads to a respective decrease or increase in exosome secretion, without altering exosome cargo content. Live-cell imaging revealed that cortactin controls both trafficking and plasma membrane docking of multivesicular late endosomes (MVEs). Regulation of exosome secretion by cortactin requires binding to the branched actin nucleating Arp2/3 complex and to actin filaments. Furthermore, cortactin, Rab27a, and coronin 1b coordinately control stability of cortical actin MVE docking sites and exosome secretion. Functionally, the addition of purified exosomes to cortactin-knockdown cells rescued defects of those cells in serum-independent growth and invasion. These data suggest a model in which cortactin promotes exosome secretion by stabilizing cortical actin-rich MVE docking sites. PMID:27402952

  1. Retinoids for prevention and treatment of actinic keratosis*

    PubMed Central

    Ianhez, Mayra; Fleury, Luiz Fernando Fróes; Miot, Hélio Amante; Bagatin, Edileia

    2013-01-01

    Actinic keratosis is a common cause of dermatological consultations and it presents a strong association with squamous cell carcinoma. Many substances are used for treatment and prevention, such as retinoids. Nevertheless, many studies on retinoids emphasize their application in treating and preventing non melanoma skin cancers. In this article, we reviewed studies about systemic and topical retinoids used with immunocompetent patients and organ transplant recipients with actinic keratosis, as primary or secondary outcomes. The majority of these papers pointed to a reduction in actinic keratosis count after treatment with retinoids. However, studies need to be better-defined in order to address the lack of a standardized dose, the absence of control groups, the low number of patients and short follow-up periods. Blind, randomized and controlled clinical trials with adequate sample sizes, specifically focused on actinic keratosis, are needed to clarify the real benefit of topical and/or oral retinoids. Comparison of efficacy and safety between oral and topical retinoids in the prevention and treatment of non-melanoma skin cancers and actinic keratosis is an essential pre requisite to establish new strategies to control these conditions. PMID:24068130

  2. Direct interaction of microtubule- and actin-based transport motors

    NASA Technical Reports Server (NTRS)

    Huang, J. D.; Brady, S. T.; Richards, B. W.; Stenolen, D.; Resau, J. H.; Copeland, N. G.; Jenkins, N. A.

    1999-01-01

    The microtubule network is thought to be used for long-range transport of cellular components in animal cells whereas the actin network is proposed to be used for short-range transport, although the mechanism(s) by which this transport is coordinated is poorly understood. For example, in sea urchins long-range Ca2+-regulated transport of exocytotic vesicles requires a microtubule-based motor, whereas an actin-based motor is used for short-range transport. In neurons, microtubule-based kinesin motor proteins are used for long-range vesicular transport but microtubules do not extend into the neuronal termini, where actin filaments form the cytoskeletal framework, and kinesins are rapidly degraded upon their arrival in neuronal termini, indicating that vesicles may have to be transferred from microtubules to actin tracks to reach their final destination. Here we show that an actin-based vesicle-transport motor, MyoVA, can interact directly with a microtubule-based transport motor, KhcU. As would be expected if these complexes were functional, they also contain kinesin light chains and the localization of MyoVA and KhcU overlaps in the cell. These results indicate that cellular transport is, in part, coordinated through the direct interaction of different motor molecules.

  3. Hippocampal Dendritic Spines Are Segregated Depending on Their Actin Polymerization

    PubMed Central

    Domínguez-Iturza, Nuria; Calvo, María; Benoist, Marion; Esteban, José Antonio; Morales, Miguel

    2016-01-01

    Dendritic spines are mushroom-shaped protrusions of the postsynaptic membrane. Spines receive the majority of glutamatergic synaptic inputs. Their morphology, dynamics, and density have been related to synaptic plasticity and learning. The main determinant of spine shape is filamentous actin. Using FRAP, we have reexamined the actin dynamics of individual spines from pyramidal hippocampal neurons, both in cultures and in hippocampal organotypic slices. Our results indicate that, in cultures, the actin mobile fraction is independently regulated at the individual spine level, and mobile fraction values do not correlate with either age or distance from the soma. The most significant factor regulating actin mobile fraction was the presence of astrocytes in the culture substrate. Spines from neurons growing in the virtual absence of astrocytes have a more stable actin cytoskeleton, while spines from neurons growing in close contact with astrocytes show a more dynamic cytoskeleton. According to their recovery time, spines were distributed into two populations with slower and faster recovery times, while spines from slice cultures were grouped into one population. Finally, employing fast lineal acquisition protocols, we confirmed the existence of loci with high polymerization rates within the spine. PMID:26881098

  4. Actin - a biosensor that determines cell fate in yeasts.

    PubMed

    Smethurst, Daniel G J; Dawes, Ian W; Gourlay, Campbell W

    2014-02-01

    The decision to proliferate, to activate stress response mechanisms or to initiate cell death lies at the heart of the maintenance of a healthy cell population. Within multicellular and colony-forming single-celled organisms, such as yeasts, the functionality of cellular compartments that connect signalling to cell fate must be maintained to maximise adaptability and survival. The actin cytoskeleton is involved in processes such as the regulation of membrane microcompartments, receptor internalisation and the control of master regulatory GTPases, which govern cell decision-making. This affords the actin cytoskeleton a central position within cell response networks. In this sense, a functional actin cytoskeleton is essential to efficiently connect information input to response at the level of the cell. Recent research from fungal, plant and mammalian cells systems has highlighted that actin can trigger apoptotic death in cells that become incompetent to respond to environmental cues. It may also be the case that this property has been appropriated by microorganisms competing for niche environments within a human host. Here, we discuss the research that has been carried out in yeast that links actin to signalling processes and cell fate that supports its role as a biosensor.

  5. Triggering signaling pathways using F-actin self-organization

    PubMed Central

    Colin, A.; Bonnemay, L.; Gayrard, C.; Gautier, J.; Gueroui, Z.

    2016-01-01

    The spatiotemporal organization of proteins within cells is essential for cell fate behavior. Although it is known that the cytoskeleton is vital for numerous cellular functions, it remains unclear how cytoskeletal activity can shape and control signaling pathways in space and time throughout the cell cytoplasm. Here we show that F-actin self-organization can trigger signaling pathways by engineering two novel properties of the microfilament self-organization: (1) the confinement of signaling proteins and (2) their scaffolding along actin polymers. Using in vitro reconstitutions of cellular functions, we found that both the confinement of nanoparticle-based signaling platforms powered by F-actin contractility and the scaffolding of engineered signaling proteins along actin microfilaments can drive a signaling switch. Using Ran-dependent microtubule nucleation, we found that F-actin dynamics promotes the robust assembly of microtubules. Our in vitro assay is a first step towards the development of novel bottom-up strategies to decipher the interplay between cytoskeleton spatial organization and signaling pathway activity. PMID:27698406

  6. Active Chemical Thermodynamics promoted by activity of cortical actin

    NASA Astrophysics Data System (ADS)

    Bhattacharya, Bhaswati; Chaudhuri, Abhishek; Gowrishankar, Kripa; Rao, Madan

    2011-03-01

    The spatial distribution and dynamics of formation and breakup of the nanoclusters of cell surface proteins is controlled by the active remodeling dynamics of the underlying cortical actin. To explain these observations, we have proposed a novel mechanism of nanoclustering, involving the transient binding to and advection along constitutively occuring ``asters'' of cortical actin. We study the consequences of such active actin-based clustering, in the context of chemical reactions involving conformational changes of cell surface proteins. We find that the active remodeling of cortical actin, can give rise to a dramatic increase in efficiency and extent of conformational spread, even at low levels of expression at the cell surface. We define a activity temperature (τa) arising due to actin activities which can be used to describe chemical thermodynamics of the system. We plot TTT (time-temparature-transformation) curves and compute the Arrhenius factors which depend on τa . With this, the active asters can be treated as enzymes whose enzymatic reaction rate can be related to the activity.

  7. Cortactin Adopts a Globular Conformation and Bundles Actin into Sheets

    SciTech Connect

    Cowieson, Nathan P.; King, Gordon; Cookson, David; Ross, Ian; Huber, Thomas; Hume, David A.; Kobe, Bostjan; Martin, Jennifer L.

    2008-08-21

    Cortactin is a filamentous actin-binding protein that plays a pivotal role in translating environmental signals into coordinated rearrangement of the cytoskeleton. The dynamic reorganization of actin in the cytoskeleton drives processes including changes in cell morphology, cell migration, and phagocytosis. In general, structural proteins of the cytoskeleton bind in the N-terminal region of cortactin and regulatory proteins in the C-terminal region. Previous structural studies have reported an extended conformation for cortactin. It is therefore unclear how cortactin facilitates cross-talk between structural proteins and their regulators. In the study presented here, circular dichroism, chemical cross-linking, and small angle x-ray scattering are used to demonstrate that cortactin adopts a globular conformation, thereby bringing distant parts of the molecule into close proximity. In addition, the actin bundling activity of cortactin is characterized, showing that fully polymerized actin filaments are bundled into sheet-like structures. We present a low resolution structure that suggests how the various domains of cortactin interact to coordinate its array of binding partners at sites of actin branching.

  8. State transitions of actin cortices in vitro and in vivo

    NASA Astrophysics Data System (ADS)

    Tan, Tzer Han; Keren, Kinneret; Mackintosh, Fred; Schmidt, Christoph; Fakhri, Nikta

    Most animal cells are enveloped by a thin layer of actin cortex which governs the cell mechanics. A functional cortex must be rigid to provide mechanical support while being flexible to allow for rapid restructuring events such as cell division. To satisfy these requirements, the actin cortex is highly dynamic with fast actin turnover and myosin-driven contractility. The regulatory mechanism responsible for the transition between a mechanically stable state and a restructuring state is not well understood. Here, we develop a technique to map the dynamics of reconstituted actin cortices in emulsion droplets using IR fluorescent single-walled carbon nanotubes (SWNTs). By increasing crosslinker concentration, we find that a homogeneous cortex transitions to an intermediate state with broken rotational symmetry and a globally contractile state which further breaks translational symmetry. We apply this new dynamic mapping technique to cortices of live starfish oocytes in various developmental stages. To identify the regulatory mechanism for steady state transitions, we subject the oocytes to actin and myosin disrupting drugs.

  9. Actin Filaments Regulate Exocytosis at the Hair Cell Ribbon Synapse.

    PubMed

    Guillet, Marie; Sendin, Gaston; Bourien, Jérôme; Puel, Jean-Luc; Nouvian, Régis

    2016-01-20

    Exocytosis at the inner hair cell ribbon synapse is achieved through the functional coupling between calcium channels and glutamate-filled synaptic vesicles. Using membrane capacitance measurements, we investigated whether the actin network regulates the exocytosis of synaptic vesicles at the mouse auditory hair cell. Our results suggest that actin network disruption increases exocytosis and that actin filaments may spatially organize a subfraction of synaptic vesicles with respect to the calcium channels. Significance statement: Inner hair cells (IHCs), the auditory sensory cells of the cochlea, release glutamate onto the afferent auditory nerve fibers to encode sound stimulation. To achieve this task, the IHC relies on the recruitment of glutamate-filled vesicles that can be located in close vicinity to the calcium channels or more remotely from them. The molecular determinants responsible for organizing these vesicle pools are not fully identified. Using pharmacological tools in combination with membrane capacitance measurements, we show that actin filament disruption increases exocytosis in IHCs and that actin filaments most likely position a fraction of vesicles away from the calcium channels. PMID:26791198

  10. 3D Actin Network Centerline Extraction with Multiple Active Contours

    PubMed Central

    Xu, Ting; Vavylonis, Dimitrios; Huang, Xiaolei

    2013-01-01

    Fluorescence microscopy is frequently used to study two and three dimensional network structures formed by cytoskeletal polymer fibers such as actin filaments and actin cables. While these cytoskeletal structures are often dilute enough to allow imaging of individual filaments or bundles of them, quantitative analysis of these images is challenging. To facilitate quantitative, reproducible and objective analysis of the image data, we propose a semi-automated method to extract actin networks and retrieve their topology in 3D. Our method uses multiple Stretching Open Active Contours (SOACs) that are automatically initialized at image intensity ridges and then evolve along the centerlines of filaments in the network. SOACs can merge, stop at junctions, and reconfigure with others to allow smooth crossing at junctions of filaments. The proposed approach is generally applicable to images of curvilinear networks with low SNR. We demonstrate its potential by extracting the centerlines of synthetic meshwork images, actin networks in 2D Total Internal Reflection Fluorescence Microscopy images, and 3D actin cable meshworks of live fission yeast cells imaged by spinning disk confocal microscopy. Quantitative evaluation of the method using synthetic images shows that for images with SNR above 5.0, the average vertex error measured by the distance between our result and ground truth is 1 voxel, and the average Hausdorff distance is below 10 voxels. PMID:24316442

  11. A Network Meta-Analysis of the Relative Efficacy of Treatments for Actinic Keratosis of the Face or Scalp in Europe

    PubMed Central

    Vegter, Stefan; Tolley, Keith

    2014-01-01

    Background Several treatments are available for actinic keratosis (AK) on the face and scalp. Most treatment modalities were compared to placebo and therefore little is known on their relative efficacy. Objectives To compare the different treatments for mild to moderate AK on the face and scalp available in clinical practice in Europe. Methods A network meta-analysis (NMA) was performed on the outcome “complete patient clearance”. Ten treatment modalities were included: two 5-aminolaevulinic acid photodynamic therapies (ALA-PDT), applied as gel (BF-200 ALA) or patch; methyl-aminolevulinate photodynamic therapy (MAL-PDT); three modalities with imiquimod (IMI), applied as a 4-week or 16-week course with 5% imiquimod, or a 2–3 week course with 3.75% imiquimod; cryotherapy; diclofenac 3% in 2.5% hyaluronic acid; 0.5% 5-fluorouracil (5-FU); and ingenol mebutate (IMB). The only data available for 5% 5-FU was from one small study and was determined to be too limited to be reliably included in the analysis. For BF-200 ALA and MAL-PDT, data from illumination with narrow-band lights were selected as these are typically used in clinical practice. The NMA was performed with a random-effects Bayesian model. Results 25 trials on 5,562 patients were included in the NMA. All active treatments were significantly better than placebo. BF-200 ALA showed the highest efficacy compared to placebo to achieve total patient clearance. BF-200 ALA had the highest probability to be the best treatment and the highest SUCRA score (64.8% and 92.1%), followed by IMI 5% 4 weeks (10.1% and 74.2%) and 5-FU 0.5% (7.2% and 66.8%). Conclusions This NMA showed that BF-200 ALA, using narrow-band lights, was the most efficacious treatment for mild to moderate AK on the face and scalp. This analysis is relevant for clinical decision making and health technology assessment, assisting the improved management of AK. PMID:24892649

  12. The Switch-associated Protein 70 (SWAP-70) Bundles Actin Filaments and Contributes to the Regulation of F-actin Dynamics*

    PubMed Central

    Chacón-Martínez, Carlos Andrés; Kiessling, Nadine; Winterhoff, Moritz; Faix, Jan; Müller-Reichert, Thomas; Jessberger, Rolf

    2013-01-01

    Coordinated assembly and disassembly of actin into filaments and higher order structures such as stress fibers and lamellipodia are fundamental for cell migration and adhesion. However, the precise spatiotemporal regulation of F-actin structures is not completely understood. SWAP-70, a phosphatidylinositol 3,4,5-trisphosphate-interacting, F-actin-binding protein, participates in actin rearrangements through yet unknown mechanisms. Here, we show that SWAP-70 is an F-actin-bundling protein that oligomerizes through a Gln/Glu-rich stretch within a coiled-coil region. SWAP-70 bundles filaments in parallel and anti-parallel fashion through its C-terminal F-actin binding domain and delays dilution-induced F-actin depolymerization. We further demonstrate that SWAP-70 co-localizes and directly interacts with cofilin, an F-actin severing and depolymerization factor, and contributes to the regulation of cofilin activity in vivo. In line with these activities, upon stem cell factor stimulation, murine bone marrow-derived mast cells lacking SWAP-70 display aberrant regulation of F-actin and actin free barbed ends dynamics. Moreover, proper stem cell factor-dependent cofilin activation via dephosphorylation and subcellular redistribution into a detergent-resistant cytoskeletal compartment also require SWAP-70. Together, these findings reveal an important role of SWAP-70 in the dynamic spatiotemporal regulation of F-actin networks. PMID:23921380

  13. Actin nucleation at the centrosome controls lymphocyte polarity

    PubMed Central

    Obino, Dorian; Farina, Francesca; Malbec, Odile; Sáez, Pablo J.; Maurin, Mathieu; Gaillard, Jérémie; Dingli, Florent; Loew, Damarys; Gautreau, Alexis; Yuseff, Maria-Isabel; Blanchoin, Laurent; Théry, Manuel; Lennon-Duménil, Ana-Maria

    2016-01-01

    Cell polarity is required for the functional specialization of many cell types including lymphocytes. A hallmark of cell polarity is the reorientation of the centrosome that allows repositioning of organelles and vesicles in an asymmetric fashion. The mechanisms underlying centrosome polarization are not fully understood. Here we found that in resting lymphocytes, centrosome-associated Arp2/3 locally nucleates F-actin, which is needed for centrosome tethering to the nucleus via the LINC complex. Upon lymphocyte activation, Arp2/3 is partially depleted from the centrosome as a result of its recruitment to the immune synapse. This leads to a reduction in F-actin nucleation at the centrosome and thereby allows its detachment from the nucleus and polarization to the synapse. Therefore, F-actin nucleation at the centrosome—regulated by the availability of the Arp2/3 complex—determines its capacity to polarize in response to external stimuli. PMID:26987298

  14. Dissecting principles governing actin assembly using yeast extracts.

    PubMed

    Michelot, Alphée; Drubin, David G

    2014-01-01

    In this chapter, we describe recent protocols that we have developed to trigger actin assembly and actin-based motility in yeast cell extracts. Our method allows for the fast preparation of yeast extracts that are competent in dynamic assembly of distinct actin filament structures of biologically appropriate protein composition. Compared to previous extract-based systems using other eukaryotic cell types, yeast provides a unique advantage for combining reconstituted assays with the preparation of extracts from genetically modified yeast strains. We present a global strategy for dissecting the functions of individual proteins, where the activities of the proteins are analyzed in systems of variable complexity, ranging from simple mixtures of pure proteins to the full complexity of a cell's cytoplasm.

  15. Formation of actin networks in microfluidic concentration gradients

    NASA Astrophysics Data System (ADS)

    Strelnikova, Natalja; Herren, Florian; Schoenenberger, Cora-Ann; Pfohl, Thomas

    2016-05-01

    The physical properties of cytoskeletal networks are contributors in a number of mechanical responses of cells including cellular deformation and locomotion, and are crucial for the proper action of living cells. Local chemical gradients modulate cytoskeletal functionality including the interactions of the cytoskeleton with other cellular components. Actin is a major constituent of the cytoskeleton. Introducing a microfluidic-based platform, we explored the impact of concentration gradients on the formation and structural properties of actin networks. Microfluidics-controlled flow-free steady state experimental conditions allow for the generation of chemical gradients of different profiles, such as linear or step-like. We discovered specific features of actin networks emerging in defined gradients. In particular, we analyzed the effects of spatial conditions on network properties, bending rigidities of network links, and the network elasticity.

  16. Involvement of actin filaments in rhizoid morphogenesis of Spirogyra.

    PubMed

    Yoshida, Katsuhisa; Shimmen, Teruo

    2009-01-01

    The role of actin filaments in rhizoid morphogenesis was studied in Spirogyra. When the algal filaments were severed, new terminal cells started tip growth and finally formed rhizoids. Actin inhibitors, latrunculin B and cytochalasin D, reversibly inhibited the process. A mesh-like structure of actin filaments (AFs) was formed at the tip region. Gd(3+) inhibited tip growth and decreased AFs in the tip region. Either a decrease in turgor pressure or lowering of the external Ca(2+) concentration also induced similar results. It was suggested that the mesh-like AF structure is indispensable for the elongation of rhizoids. A possible organization mechanism of the mesh-like AF structure was discussed.

  17. Formin-mediated actin polymerization promotes Salmonella invasion.

    PubMed

    Truong, Dorothy; Brabant, Danielle; Bashkurov, Mikhail; Wan, Leo C K; Braun, Virginie; Heo, Won Do; Meyer, Tobias; Pelletier, Laurence; Copeland, John; Brumell, John H

    2013-12-01

    Salmonella invade host cells using Type 3 secreted effectors, which modulate host cellular targets to promote actin rearrangements at the cell surface that drive bacterial uptake. The Arp2/3 complex contributes to Salmonella invasion but is not essential, indicating other actin regulatory factors are involved. Here, we show a novel role for FHOD1, a formin family member, in Salmonella invasion. FHOD1 and Arp2/3 occupy distinct microdomains at the invasion site and control distinct aspects of membrane protrusion formation. FHOD1 is phosphorylated during infection and this modification is required for promoting bacterial uptake by host cells. ROCK II, but not ROCK I, is recruited to the invasion site and is required for FHOD1 phosphorylation and for Salmonella invasion. Together, our studies revealan important phospho-dependent FHOD1 actin polymerization pathway in Salmonella invasion.

  18. The interest of actin immunocytochemistry in diagnostic histopathology.

    PubMed

    Bussolati, G; Papotti, M; Foschini, M P; Eusebi, V

    1987-01-01

    The immunohistochemical detection of different isoforms of actin provides useful data in the histopathological diagnosis of human tumours. The marked resistance of this protein to fixation and embedding procedures makes it a practical alternative to the ultrastructural search of microfilaments in routine tissue sections. Staining with monoclonal antibodies against alpha smooth muscle actin is helpful in the diagnosis of benign and malignant smooth muscle tumours and allows the differentiation of various types of spindle cell sarcomas. These antibodies are also useful in tracing the myoepithelial cells in normal and various pathological conditions of the breast and salivary glands. Skeletal muscle actin is a reliable marker in the diagnosis of rhabdomyosarcomas. Its use should be combined and complemented with other markers (i.e. desmin; foetal, slow and fast myosins; myoglobin) to monitor the degree of rhabdomyoblastic differentiation of the neoplastic cells in single cases, a parameter of potential prognostic value.

  19. Nicotiana tabacum actin-depolymerizing factor 2 is involved in actin-driven, auxin-dependent patterning.

    PubMed

    Durst, Steffen; Nick, Peter; Maisch, Jan

    2013-08-15

    Polar transport of auxin has been identified as a central element of pattern formation. To address the underlying cellular mechanisms, we use the tobacco cell line (Nicotiana tabacum L. cv. Bright Yellow 2; BY-2) as model. We showed previously that cell divisions within a cell file are synchronized by polar auxin flow, linked to the organization of actin filaments (AF) which, in turn, is modified via actin-binding proteins (ABPs). From a preparatory study for disturbed division synchrony in cell lines overexpressing different ABPs, we identified the actin depolymerizing factor 2 (ADF2). A cell line overexpressing GFP-NtADF2 was specifically affected in division synchrony. The cell division pattern could be rescued by addition of Phosphatidylinositol 4,5-bisphosphate (PIP2) or by phalloidin. These observations allow to draw first conclusions on the pathway linking auxin signalling via actin reorganization to synchronized cell division placing the regulation of cortical actin turnover by ADF2 into the focus. PMID:23545293

  20. The association of myosin IB with actin waves in dictyostelium requires both the plasma membrane-binding site and actin-binding region in the myosin tail.

    PubMed

    Brzeska, Hanna; Pridham, Kevin; Chery, Godefroy; Titus, Margaret A; Korn, Edward D

    2014-01-01

    F-actin structures and their distribution are important determinants of the dynamic shapes and functions of eukaryotic cells. Actin waves are F-actin formations that move along the ventral cell membrane driven by actin polymerization. Dictyostelium myosin IB is associated with actin waves but its role in the wave is unknown. Myosin IB is a monomeric, non-filamentous myosin with a globular head that binds to F-actin and has motor activity, and a non-helical tail comprising a basic region, a glycine-proline-glutamine-rich region and an SH3-domain. The basic region binds to acidic phospholipids in the plasma membrane through a short basic-hydrophobic site and the Gly-Pro-Gln region binds F-actin. In the current work we found that both the basic-hydrophobic site in the basic region and the Gly-Pro-Gln region of the tail are required for the association of myosin IB with actin waves. This is the first evidence that the Gly-Pro-Gln region is required for localization of myosin IB to a specific actin structure in situ. The head is not required for myosin IB association with actin waves but binding of the head to F-actin strengthens the association of myosin IB with waves and stabilizes waves. Neither the SH3-domain nor motor activity is required for association of myosin IB with actin waves. We conclude that myosin IB contributes to anchoring actin waves to the plasma membranes by binding of the basic-hydrophobic site to acidic phospholipids in the plasma membrane and binding of the Gly-Pro-Gln region to F-actin in the wave.

  1. Computational Tension Mapping of Adherent Cells Based on Actin Imaging

    PubMed Central

    Manifacier, Ian; Milan, Jean-Louis; Jeanneau, Charlotte; Chmilewsky, Fanny; Chabrand, Patrick; About, Imad

    2016-01-01

    Forces transiting through the cytoskeleton are known to play a role in adherent cell activity. Up to now few approaches haves been able to determine theses intracellular forces. We thus developed a computational mechanical model based on a reconstruction of the cytoskeleton of an adherent cell from fluorescence staining of the actin network and focal adhesions (FA). Our custom made algorithm converted the 2D image of an actin network into a map of contractile interactions inside a 2D node grid, each node representing a group of pixels. We assumed that actin filaments observed under fluorescence microscopy, appear brighter when thicker, we thus presumed that nodes corresponding to pixels with higher actin density were linked by stiffer interactions. This enabled us to create a system of heterogeneous interactions which represent the spatial organization of the contractile actin network. The contractility of this interaction system was then adapted to match the level of force the cell truly exerted on focal adhesions; forces on focal adhesions were estimated from their vinculin expressed size. This enabled the model to compute consistent mechanical forces transiting throughout the cell. After computation, we applied a graphical approach on the original actin image, which enabled us to calculate tension forces throughout the cell, or in a particular region or even in single stress fibers. It also enabled us to study different scenarios which may indicate the mechanical role of other cytoskeletal components such as microtubules. For instance, our results stated that the ratio between intra and extra cellular compression is inversely proportional to intracellular tension. PMID:26812601

  2. Microtubule and Actin Interplay Drive Intracellular c-Src Trafficking.

    PubMed

    Arnette, Christopher; Frye, Keyada; Kaverina, Irina

    2016-01-01

    The proto-oncogene c-Src is involved in a variety of signaling processes. Therefore, c-Src spatiotemporal localization is critical for interaction with downstream targets. However, the mechanisms regulating this localization have remained elusive. Previous studies have shown that c-Src trafficking is a microtubule-dependent process that facilitates c-Src turnover in neuronal growth cones. As such, microtubule depolymerization lead to the inhibition of c-Src recycling. Alternatively, c-Src trafficking was also shown to be regulated by RhoB-dependent actin polymerization. Our results show that c-Src vesicles primarily exhibit microtubule-dependent trafficking; however, microtubule depolymerization does not inhibit vesicle movement. Instead, vesicular movement becomes both faster and less directional. This movement was associated with actin polymerization directly at c-Src vesicle membranes. Interestingly, it has been shown previously that c-Src delivery is an actin polymerization-dependent process that relies on small GTPase RhoB at c-Src vesicles. In agreement with this finding, microtubule depolymerization induced significant activation of RhoB, together with actin comet tail formation. These effects occurred downstream of GTP-exchange factor, GEF-H1, which was released from depolymerizing MTs. Accordingly, GEF-H1 activity was necessary for actin comet tail formation at the Src vesicles. Our results indicate that regulation of c-Src trafficking requires both microtubules and actin polymerization, and that GEF-H1 coordinates c-Src trafficking, acting as a molecular switch between these two mechanisms. PMID:26866809

  3. Identification of Actin-Binding Proteins from Maize Pollen

    SciTech Connect

    Staiger, C.J.

    2004-01-13

    Specific Aims--The goal of this project was to gain an understanding of how actin filament organization and dynamics are controlled in flowering plants. Specifically, we proposed to identify unique proteins with novel functions by investigating biochemical strategies for the isolation and characterization of actin-binding proteins (ABPs). In particular, our hunt was designed to identify capping proteins and nucleation factors. The specific aims included: (1) to use F-actin affinity chromatography (FAAC) as a general strategy to isolate pollen ABPs (2) to produce polyclonal antisera and perform subcellular localization in pollen tubes (3) to isolate cDNA clones for the most promising ABPs (4) to further purify and characterize ABP interactions with actin in vitro. Summary of Progress By employing affinity chromatography on F-actin or DNase I columns, we have identified at least two novel ABPs from pollen, PrABP80 (gelsolin-like) and ZmABP30, We have also cloned and expressed recombinant protein, as well as generated polyclonal antisera, for 6 interesting ABPs from Arabidopsis (fimbrin AtFIM1, capping protein a/b (AtCP), adenylyl cyclase-associated protein (AtCAP), AtCapG & AtVLN1). We performed quantitative analyses of the biochemical properties for two of these previously uncharacterized ABPs (fimbrin and capping protein). Our studies provide the first evidence for fimbrin activity in plants, demonstrate the existence of barbed-end capping factors and a gelsolin-like severing activity, and provide the quantitative data necessary to establish and test models of F-actin organization and dynamics in plant cells.

  4. Microtubule and Actin Interplay Drive Intracellular c-Src Trafficking

    PubMed Central

    Arnette, Christopher; Frye, Keyada; Kaverina, Irina

    2016-01-01

    The proto-oncogene c-Src is involved in a variety of signaling processes. Therefore, c-Src spatiotemporal localization is critical for interaction with downstream targets. However, the mechanisms regulating this localization have remained elusive. Previous studies have shown that c-Src trafficking is a microtubule-dependent process that facilitates c-Src turnover in neuronal growth cones. As such, microtubule depolymerization lead to the inhibition of c-Src recycling. Alternatively, c-Src trafficking was also shown to be regulated by RhoB-dependent actin polymerization. Our results show that c-Src vesicles primarily exhibit microtubule-dependent trafficking; however, microtubule depolymerization does not inhibit vesicle movement. Instead, vesicular movement becomes both faster and less directional. This movement was associated with actin polymerization directly at c-Src vesicle membranes. Interestingly, it has been shown previously that c-Src delivery is an actin polymerization-dependent process that relies on small GTPase RhoB at c-Src vesicles. In agreement with this finding, microtubule depolymerization induced significant activation of RhoB, together with actin comet tail formation. These effects occurred downstream of GTP-exchange factor, GEF-H1, which was released from depolymerizing MTs. Accordingly, GEF-H1 activity was necessary for actin comet tail formation at the Src vesicles. Our results indicate that regulation of c-Src trafficking requires both microtubules and actin polymerization, and that GEF-H1 coordinates c-Src trafficking, acting as a molecular switch between these two mechanisms. PMID:26866809

  5. Fluorescent labelling of the actin cytoskeleton in plants using a cameloid antibody

    PubMed Central

    2014-01-01

    Background Certain members of the Camelidae family produce a special type of antibody with only one heavy chain. The antigen binding domains are the smallest functional fragments of these heavy-chain only antibodies and as a consequence have been termed nanobodies. Discovery of these nanobodies has allowed the development of a number of therapeutic proteins and tools. In this study a class of nanobodies fused to fluorescent proteins (chromobodies), and therefore allowing antigen-binding and visualisation by fluorescence, have been used. Such chromobodies can be expressed in living cells and used as genetically encoded immunocytochemical markers. Results Here a modified version of the commercially available Actin-Chromobody® as a novel tool for visualising actin dynamics in tobacco leaf cells was tested. The actin-chromobody binds to actin in a specific manner. Treatment with latrunculin B, a drug which disrupts the actin cytoskeleton through inhibition of polymerisation results in loss of fluorescence after less than 30 min but this can be rapidly restored by washing out latrunculin B and thereby allowing the actin filaments to repolymerise. To test the effect of the actin-chromobody on actin dynamics and compare it to one of the conventional labelling probes, Lifeact, the effect of both probes on Golgi movement was studied as the motility of Golgi bodies is largely dependent on the actin cytoskeleton. With the actin-chromobody expressed in cells, Golgi body movement was slowed down but the manner of movement rather than speed was affected less than with Lifeact. Conclusions The actin-chromobody technique presented in this study provides a novel option for in vivo labelling of the actin cytoskeleton in comparison to conventionally used probes that are based on actin binding proteins. The actin-chromobody is particularly beneficial to study actin dynamics in plant cells as it does label actin without impairing dynamic movement and polymerisation of the actin

  6. Actin and Arp2/3 localize at the centrosome of interphase cells

    SciTech Connect

    Hubert, Thomas; Vandekerckhove, Joel; Gettemans, Jan

    2011-01-07

    Research highlights: {yields} Actin was detected at the centrosome with the anti-actin antibody 1C7 that recognizes antiparallel ('lower dimer') actin dimers. {yields} Centrosomal actin was found in interphase but not mitotic MDA-MB-231 cells. {yields} Neither the anti-actin antibody C4 that binds to globular, monomer actin, nor the anti-actin antibody 2G2 that recognizes the nuclear conformation of actin detect actin at the centrosome. {yields} The Arp2/3 complex transiently localizes at the pericentriolar matrix but not at the centrioles of interphase HEK 293T cells. -- Abstract: Although many actin binding proteins such as cortactin and the Arp2/3 activator WASH localize at the centrosome, the presence and conformation of actin at the centrosome has remained elusive. Here, we report the localization of actin at the centrosome in interphase but not in mitotic MDA-MB-231 cells. Centrosomal actin was detected with the anti-actin antibody 1C7 that recognizes antiparallel ('lower dimer') actin dimers. In addition, we report the transient presence of the Arp2/3 complex at the pericentriolar matrix but not at the centrioles of interphase HEK 293T cells. Overexpression of an Arp2/3 component resulted in expansion of the pericentriolar matrix and selective accumulation of the Arp2/3 component in the pericentriolar matrix. Altogether, we hypothesize that the centrosome transiently recruits Arp2/3 to perform processes such as centrosome separation prior to mitotic entry, whereas the observed constitutive centrosomal actin staining in interphase cells reinforces the current model of actin-based centrosome reorientation toward the leading edge in migrating cells.

  7. Two Functionally Distinct Sources of Actin Monomers Supply the Leading Edge of Lamellipodia

    PubMed Central

    Vitriol, Eric A.; McMillen, Laura M.; Kapustina, Maryna; Gomez, Shawn M.; Vavylonis, Dimitrios; Zheng, James Q.

    2015-01-01

    Summary Lamellipodia, the sheet-like protrusions of motile cells, consist of networks of actin filaments (F-actin) regulated by the ordered assembly from and disassembly into actin monomers (G-actin). Traditionally, G-actin is thought to exist as a homogeneous pool. Here, we show that there are two functionally and molecularly distinct sources of G-actin that supply lamellipodial actin networks. G-actin originating from the cytosolic pool requires the monomer binding protein thymosin β4 (Tβ4) for optimal leading edge localization, is targeted to formins, and is responsible for creating an elevated G/F-actin ratio that promotes membrane protrusion. The second source of G-actin comes from recycled lamellipodia F-actin. Recycling occurs independently of Tβ4 and appears to regulate lamellipodia homeostasis. Tβ4-bound G-actin specifically localizes to the leading edge because it doesn’t interact with Arp2/3-mediated polymerization sites found throughout the lamellipodia. These findings demonstrate that actin networks can be constructed from multiple sources of monomers with discrete spatiotemporal functions. PMID:25865895

  8. Mechanical stimulation induces formin-dependent assembly of a perinuclear actin rim

    PubMed Central

    Shao, Xiaowei; Li, Qingsen; Mogilner, Alex; Bershadsky, Alexander D.; Shivashankar, G. V.

    2015-01-01

    Cells constantly sense and respond to mechanical signals by reorganizing their actin cytoskeleton. Although a number of studies have explored the effects of mechanical stimuli on actin dynamics, the immediate response of actin after force application has not been studied. We designed a method to monitor the spatiotemporal reorganization of actin after cell stimulation by local force application. We found that force could induce transient actin accumulation in the perinuclear region within ∼2 min. This actin reorganization was triggered by an intracellular Ca2+ burst induced by force application. Treatment with the calcium ionophore A23187 recapitulated the force-induced perinuclear actin remodeling. Blocking of actin polymerization abolished this process. Overexpression of Klarsicht, ANC-1, Syne Homology (KASH) domain to displace nesprins from the nuclear envelope did not abolish Ca2+-dependent perinuclear actin assembly. However, the endoplasmic reticulum- and nuclear membrane-associated inverted formin-2 (INF2), a potent actin polymerization activator (mutations of which are associated with several genetic diseases), was found to be important for perinuclear actin assembly. The perinuclear actin rim structure colocalized with INF2 on stimulation, and INF2 depletion resulted in attenuation of the rim formation. Our study suggests that cells can respond rapidly to external force by remodeling perinuclear actin in a unique Ca2+- and INF2-dependent manner. PMID:25941386

  9. How capping protein enhances actin filament growth and nucleation on biomimetic beads

    NASA Astrophysics Data System (ADS)

    Wang, Ruizhe; Carlsson, Anders E.

    2015-12-01

    Capping protein (CP), which caps the growing ends of actin filaments, accelerates actin-based motility. Recent experiments on biomimetic beads have shown that CP also enhances the rate of actin filament nucleation. Proposed explanations for these phenomena include (i) the actin funneling hypothesis (AFH), in which the presence of CP increases the free-actin concentration, and (ii) the monomer gating model, in which CP binding to actin filament barbed ends makes more monomers available for filament nucleation. To establish how CP increases the rates of filament elongation and nucleation on biomimetic beads, we perform a quantitative modeling analysis of actin polymerization, using rate equations that include actin filament nucleation, polymerization and capping, as modified by monomer depletion near the surface of the bead. With one adjustable parameter, our simulation results match previously measured time courses of polymerized actin and filament number. The results support a version of the AFH where CP increases the local actin monomer concentration at the bead surface, but leaves the global free-actin concentration nearly constant. Because the rate of filament nucleation increases with the monomer concentration, the increased local monomer concentration enhances actin filament nucleation. We derive a closed-form formula for the characteristic CP concentration where the local free-actin concentration reaches half the bulk value, and find it to be comparable to the global Arp2/3 complex concentration. We also propose an experimental protocol for distinguishing branching nucleation of filaments from spontaneous nucleation.

  10. 75 FR 62459 - Revision of Class E Airspace; Unalakleet, AK

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-10-12

    ... for air traffic management of Instrument Flight Rules (IFR) operations. DATES: Effective 0901 UTC... Federal Register to revise Class E airspace at Unalakleet, AK (75 FR 32865). Interested parties were... amendments to existing SIAPs require that the orientation and dimensions of Class E airspace be revised...

  11. 75 FR 76294 - Radio Broadcasting Services; Fairbanks, AK

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-12-08

    ... COMMISSION 47 CFR Part 73 Radio Broadcasting Services; Fairbanks, AK AGENCY: Federal Communications... Review Act, see U.S.C. 801(a)(1)(A). List of Subjects in 47 CFR Part 73 Radio, Radio broadcasting. 0 For... follows: PART 73--RADIO BROADCAST SERVICES 0 1. The authority citation for Part 73 continues to read...

  12. 76 FR 8626 - Revision of Class E Airspace; Shungnak, AK

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-02-15

    ... U.S.C. 106(g), 40103, 40113, 40120; E.O. 10854, 24 FR 9565, 3 CFR, 1959-1963 Comp., p. 389. Sec. 71... airspace at Shungnak, AK (75 FR 77573). Interested parties were invited to participate in this rulemaking... ``significant rule'' under DOT Regulatory Policies and Procedures (44 FR 11034; February 26, 1979); and (3)...

  13. Ak-Chin Indian Community Biomass Feasiiblity Study

    SciTech Connect

    Mark A. Moser, RCM Digesters, Inc.; Mark Randall, Daystar Consulting, LLC; Leonard S. Gold, Ak-Chin Energy Services & Utility Strategies Consulting Group

    2005-12-31

    Study of the conversion of chicken litter to biogas for the production of energy. There was an additional requirement that after extracting the energy from the chicken litter the nutrient value of the raw chicken litter had to be returned to the Ak-Chin Farms for use as fertilizer in a form and delivery method acceptable to the Farm.

  14. 78 FR 8962 - Establishment of Class E Airspace; Kasigluk, AK

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-02-07

    ... airspace at Kasigluk, AK, to accommodate aircraft using Area Navigation (RNAV) Global Positioning System... FURTHER INFORMATION CONTACT: Richard Roberts, Federal Aviation Administration, Operations Support Group... INFORMATION: History On October 4, 2012, the FAA published in the Federal Register a notice of...

  15. 76 FR 75447 - Amendment of Class E Airspace; Emmonak, AK

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-02

    ... Federal Register a notice of proposed rulemaking (NPRM) to amend controlled airspace at Emmonak, AK (76 FR... 12866; (2) is not a ``significant rule'' under DOT Regulatory Policies and Procedures (44 FR 11034... FR 9565, 3 CFR, 1959-1963 Comp., p. 389. Sec. 71.1 0 2. The incorporation by reference in 14 CFR...

  16. 76 FR 43824 - Revision of Class E Airspace; Talkeetna, AK

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-07-22

    ... revise Class E airspace at Talkeetna, AK (76 FR 27619). Interested parties were invited to participate in... U.S.C. 106(g), 40103, 40113, 40120; E.O. 10854, 24 FR 9565, 3 CFR, 1959-1963 Comp., p. 389. Sec. 71... not a ] ``significant rule'' under DOT Regulatory Policies and Procedures (44 FR 11034; February...

  17. 76 FR 43823 - Revision of Class E Airspace; Yakutat, AK

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-07-22

    ... U.S.C. 106(g), 40103, 40113, 40120; E.O. 10854, 24 FR 9565, 3 CFR, 1959-1963 Comp., p. 389. Sec. 71... airspace to accommodate new SIAPS at Yakutat Airport, Yakutat, AK (76 FR 21832). Interested parties were... ``significant rule'' under DOT Regulatory Policies and Procedures (44 FR 11034; February 26, 1979); and (3)...

  18. 76 FR 65944 - Establishment of Class E Airspace; Tatitlek, AK

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-10-25

    ... to establish Class E airspace at Tatitlek, AK (76 FR 49388). Interested parties were invited to... U.S.C. 106(g), 40103, 40113, 40120; E.O. 10854, 24 FR 9565, 3 CFR, 1959-1963 Comp., p. 389. Sec. 71... not a ``significant rule'' under DOT Regulatory Policies and Procedures (44 FR 11034; February...

  19. 76 FR 66854 - Amendment of Class E Airspace; Nuiqsut, AK

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-10-28

    ... rulemaking (NPRM) in the Federal Register to revise Class E airspace at Nuiqsut, AK (76 FR 49386). Interested..., 40113, 40120; E.O. 10854, 24 FR 9565, 3 CFR, 1959-1963 Comp., p. 389. Sec. 71.1 0 2. The incorporation... ``significant rule'' under DOT Regulatory Policies and Procedures (44 FR 11034; February 26, 1979); and (3)...

  20. 76 FR 66178 - Revision of Class E Airspace; Umiat, AK

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-10-26

    ... proposed rulemaking (NPRM) in the Federal Register to revise Class E airspace at Umiat, AK (76 FR 49387... Procedures (44 FR 11034; February 26, 1979); and (3) does not warrant preparation of a regulatory evaluation.... 10854, 24 FR 9565, 3 CFR, 1959-1963 Comp., p. 389. Sec. 71.1 0 2. The incorporation by reference in...

  1. 78 FR 45849 - Amendment of Class E Airspace; Gustavus, AK

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-07-30

    ... Register a notice of proposed rulemaking (NPRM) to amend controlled airspace at Gustavus, AK (78 FR 31871... 12866; (2) is not a ``significant rule'' under DOT Regulatory Policies and Procedures (44 FR 11034...), 40103, 40113, 40120; E.O. 10854, 24 FR 9565, 3 CFR, 1959-1963 Comp., p. 389. Sec. 71.1 0 2....

  2. 78 FR 5128 - Amendment of Class E Airspace; Savoonga, AK

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-01-24

    ... modify controlled airspace at Savoonga, AK (77 FR 61304). Interested parties were invited to participate... Regulatory Policies and Procedures (44 FR 11034; February 26, 1979); and (3) does not warrant preparation of... read as follows: Authority: 49 U.S.C. 106(g), 40103, 40113, 40120; E. O. 10854, 24 FR 9565, 3 CFR,...

  3. 78 FR 59622 - Establishment of Class E Airspace; Akutan, AK

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-09-27

    ..., Akutan, AK (78 FR 45477). Interested parties were invited to participate in this rulemaking effort by... Executive Order 12866; (2) is not a ``significant rule'' under DOT Regulatory Policies and Procedures (44 FR...), 40103, 40113, 40120; E.O. 10854, 24 FR 9565, 3 CFR, 1959-1963 Comp., p. 389. Sec. 71.1 0 2....

  4. 78 FR 16399 - Amendment of Class E Airspace; Unalakleet, AK

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-03-15

    ... (NPRM) to modify controlled airspace at Unalakleet, AK (77 FR 27149). Interested parties were invited to... Policies and Procedures (44 FR 11034; February 26, 1979); and (3) does not warrant preparation of a... read as follows: Authority: 49 U.S.C. 106(g), 40103, 40113, 40120; E. O. 10854, 24 FR 9565, 3 CFR,...

  5. 76 FR 8627 - Revision of Class E Airspace; Platinum, AK

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-02-15

    ... Class E airspace at Platinum AK (75 FR 77572). Interested parties were invited to participate in this... ``significant rule'' under DOT Regulatory Policies and Procedures (44 FR 11034; February 26, 1979); and (3) does... U.S.C. 106(g), 40103, 40113, 40120; E.O. 10854, 24 FR 9565, 3 CFR, 1959-1963 Comp., p. 389. Sec....

  6. 76 FR 60714 - Revision of Class E Airspace; Northway, AK

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-09-30

    ... proposed rulemaking (NPRM) in the Federal Register to revise Class E airspace at Northway, AK (76 FR 45475...: Authority: 49 U.S.C. 106(g), 40103, 40113, 40120; E.O. 10854, 24 FR 9565, 3 CFR, 1959-1963 Comp., p. 389...; (2) is not a ``significant rule'' under DOT Regulatory Policies and Procedures (44 FR 11034;...

  7. 75 FR 12677 - Establishment of Class E Airspace; Shaktoolik, AK

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-17

    ...-7587; telephone number (907) 271-5898; fax: (907) 271-2850; e-mail: gary.ctr.rolf@faa.gov . Internet.../rulemaking/ . SUPPLEMENTARY INFORMATION: History On Wednesday, December 16, 2009, the FAA published a notice of proposed rulemaking in the Federal Register to establish Class E airspace at Shaktoolik, AK (74...

  8. 75 FR 12676 - Establishment of Class E Airspace; Koyukuk, AK

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-17

    ... number (907) 271-5898; fax: (907) 271-2850; e-mail: gary.ctr.rolf@faa.gov . Internet address: http://www.../ . SUPPLEMENTARY INFORMATION: History On Wednesday, December 16, 2009, the FAA published a notice of proposed rulemaking in the Federal Register to establish Class E airspace at Koyukuk, AK (74 FR 66594)....

  9. 75 FR 32269 - Revision of Class E Airspace; Wainwright, AK

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-06-08

    ... rulemaking in the Federal Register to revise Class E airspace at Wainwright, AK (75 FR 11480). Interested...; (2) is not a ``significant rule'' under DOT Regulatory Policies and Procedures (44 FR 11034; February... FR 9565, 3 CFR, 1959-1963 Comp., p. 389. Sec. 71.1 0 2. The incorporation by reference in 14 CFR...

  10. 77 FR 6463 - Amendment of Class E Airspace; Kwigillingok, AK

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-08

    ...;having general applicability and legal effect, most of which are keyed #0;to and codified in the Code of...-2011-0881 was published in the Federal Register (77 FR 6), amending controlled airspace at Kwigillingok Airport, AK. Subsequent to publication, an error was found in the latitude coordinate for...

  11. 75 FR 33165 - Revision of Class E Airspace; Galena, AK

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-06-11

    ... FR 9565, 3 CFR, 1959-1963 Comp., p. 389. Sec. 71.1 0 2. The incorporation by reference in 14 CFR 71.1... airspace at Galena, AK (75 FR 17892). Interested parties were invited to participate in this rulemaking... Procedures (44 FR 11034; February 26, 1979); and (3) does not warrant preparation of a regulatory...

  12. 77 FR 6 - Amendment of Class E Airspace; Kwigillingok, AK

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-01-03

    ... controlled airspace at Kwigillingok, AK (76 FR 54151). Interested parties were invited to participate in this... Regulatory Policies and Procedures (44 FR 11034; February 26, 1979); and (3) does not warrant preparation of... follows: Authority: 49 U.S.C. 106(g), 40103, 40113, 40120; E.O. 10854, 24 FR 9565, 3 CFR, 1959-1963...

  13. 78 FR 4586 - Environmental Impact Statement: Kake Access, AK

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-01-22

    ... Federal Highway Administration Environmental Impact Statement: Kake Access, AK ACTION: Notice of intent... issuing this notice to advise the public that FHWA will prepare an Environmental Impact Statement (EIS... Meetings will be held in the early Spring of 2013 to receive oral and written comments on...

  14. 76 FR 8625 - Revision of Class E Airspace; Savoonga, AK

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-02-15

    ... Federal Register to amend Class E airspace at Savoonga, AK (75 FR 77574). Interested parties were invited... Norton Sound Low Offshore Airspace Area and that airspace will be redefined in a future rulemaking action... not a ``significant rule'' under DOT Regulatory Policies and Procedures (44 FR 11034; February...

  15. 75 FR 32272 - Revision of Class E Airspace; Kaltag, AK

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-06-08

    .../headquarters_offices/ato/service_units/systemops/fs/alaskan/rulemaking/ . SUPPLEMENTARY INFORMATION: History On... revise Class E airspace at Kaltag, AK (75 FR 11479). Interested parties were invited to participate in... Regulatory Policies and Procedures (44 FR 11034; February 26, 1979); and (3) does not warrant preparation...

  16. 76 FR 61258 - Revision of Class E Airspace; Allakaket, AK

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-10-04

    ..._offices/ato/service_units/systemops/fs/alaskan/rulemaking/ . SUPPLEMENTARY INFORMATION: History On Friday... revise Class E airspace at Allakaket, AK (76 FR 45477). Interested parties were invited to participate in... not a ``significant rule'' under DOT ] Regulatory Policies and Procedures (44 FR 11034; February...

  17. 75 FR 62458 - Revision of Class E Airspace; Tanana, AK

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-10-12

    .../office_org/headquarters_offices/ato/service_units/systemops/fs/alaskan/rulemaking/ . SUPPLEMENTARY... Federal Register to revise Class E airspace at the Ralph M. Calhoun Memorial Airport, Tanana, AK (75 FR... not a ``significant rule'' under DOT Regulatory Policies and Procedures (44 FR 11034; February...

  18. 75 FR 32271 - Revision of Class E Airspace; Nenana, AK

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-06-08

    ... address: http://www.faa.gov/about/office_org/headquarters_offices/ato/service_units/systemops/fs/alaskan... proposed rulemaking in the Federal Register to revise Class E airspace at Nenana, AK (75 FR 11481... Procedures (44 FR 11034; February 26, 1979); and (3) does not warrant preparation of a regulatory...

  19. Dynamic actin cycling through mitochondrial subpopulations locally regulates the fission–fusion balance within mitochondrial networks

    PubMed Central

    Moore, Andrew S.; Wong, Yvette C.; Simpson, Cory L.; Holzbaur, Erika L. F.

    2016-01-01

    Mitochondria form interconnected networks that dynamically remodel in response to cellular needs. Using live-cell imaging, we investigate the role of the actin cytoskeleton in regulating mitochondrial fission and fusion. We identify cycling of actin filaments onto and off of subsets of cellular mitochondria. The association of actin filaments with mitochondrial subpopulations is transient; actin quickly disassembles, then reassembles around a distinct subpopulation, efficiently cycling through all cellular mitochondria within 14 min. The focal assembly of actin induces local, Drp1-dependent fragmentation of the mitochondrial network. On actin disassembly, fragmented mitochondria undergo rapid fusion, leading to regional recovery of the tubular mitochondrial network. Cycling requires dynamic actin polymerization and is blocked by inhibitors of both Arp2/3 and formins. We propose that cyclic assembly of actin onto mitochondria modulates the fission/fusion balance, promotes network remodelling and content mixing, and thus may serve as an essential mechanism regulating mitochondrial network homeostasis. PMID:27686185

  20. Fimbrin phosphorylation by metaphase Cdk1 regulates actin cable dynamics in budding yeast.

    PubMed

    Miao, Yansong; Han, Xuemei; Zheng, Liangzhen; Xie, Ying; Mu, Yuguang; Yates, John R; Drubin, David G

    2016-01-01

    Actin cables, composed of actin filament bundles nucleated by formins, mediate intracellular transport for cell polarity establishment and maintenance. We previously observed that metaphase cells preferentially promote actin cable assembly through cyclin-dependent kinase 1 (Cdk1) activity. However, the relevant metaphase Cdk1 targets were not known. Here we show that the highly conserved actin filament crosslinking protein fimbrin is a critical Cdk1 target for actin cable assembly regulation in budding yeast. Fimbrin is specifically phosphorylated on threonine 103 by the metaphase cyclin-Cdk1 complex, in vivo and in vitro. On the basis of conformational simulations, we suggest that this phosphorylation stabilizes fimbrin's N-terminal domain, and modulates actin filament binding to regulate actin cable assembly and stability in cells. Overall, this work identifies fimbrin as a key target for cell cycle regulation of actin cable assembly in budding yeast, and suggests an underlying mechanism.

  1. Fimbrin phosphorylation by metaphase Cdk1 regulates actin cable dynamics in budding yeast.

    PubMed

    Miao, Yansong; Han, Xuemei; Zheng, Liangzhen; Xie, Ying; Mu, Yuguang; Yates, John R; Drubin, David G

    2016-01-01

    Actin cables, composed of actin filament bundles nucleated by formins, mediate intracellular transport for cell polarity establishment and maintenance. We previously observed that metaphase cells preferentially promote actin cable assembly through cyclin-dependent kinase 1 (Cdk1) activity. However, the relevant metaphase Cdk1 targets were not known. Here we show that the highly conserved actin filament crosslinking protein fimbrin is a critical Cdk1 target for actin cable assembly regulation in budding yeast. Fimbrin is specifically phosphorylated on threonine 103 by the metaphase cyclin-Cdk1 complex, in vivo and in vitro. On the basis of conformational simulations, we suggest that this phosphorylation stabilizes fimbrin's N-terminal domain, and modulates actin filament binding to regulate actin cable assembly and stability in cells. Overall, this work identifies fimbrin as a key target for cell cycle regulation of actin cable assembly in budding yeast, and suggests an underlying mechanism. PMID:27068241

  2. Fimbrin phosphorylation by metaphase Cdk1 regulates actin cable dynamics in budding yeast

    PubMed Central

    Miao, Yansong; Han, Xuemei; Zheng, Liangzhen; Xie, Ying; Mu, Yuguang; Yates, John R.; Drubin, David G.

    2016-01-01

    Actin cables, composed of actin filament bundles nucleated by formins, mediate intracellular transport for cell polarity establishment and maintenance. We previously observed that metaphase cells preferentially promote actin cable assembly through cyclin-dependent kinase 1 (Cdk1) activity. However, the relevant metaphase Cdk1 targets were not known. Here we show that the highly conserved actin filament crosslinking protein fimbrin is a critical Cdk1 target for actin cable assembly regulation in budding yeast. Fimbrin is specifically phosphorylated on threonine 103 by the metaphase cyclin–Cdk1 complex, in vivo and in vitro. On the basis of conformational simulations, we suggest that this phosphorylation stabilizes fimbrin's N-terminal domain, and modulates actin filament binding to regulate actin cable assembly and stability in cells. Overall, this work identifies fimbrin as a key target for cell cycle regulation of actin cable assembly in budding yeast, and suggests an underlying mechanism. PMID:27068241

  3. Actinic lichen planus mimicking melasma. A clinical and histopathologic study of three cases.

    PubMed

    Salman, S M; Khallouf, R; Zaynoun, S

    1988-02-01

    Three cases of actinic lichen planus mimicking melasma are presented. Although the clinical similarity to melasma is striking, the histopathologic picture is distinctive. It is suggested that actinic lichen planus be considered in the differential diagnosis of melasma.

  4. Arabidopsis Actin Depolymerizing Factor4 Modulates the Stochastic Dynamic Behavior of Actin Filaments in the Cortical Array of Epidermal Cells[C][W

    PubMed Central

    Henty, Jessica L.; Bledsoe, Samuel W.; Khurana, Parul; Meagher, Richard B.; Day, Brad; Blanchoin, Laurent; Staiger, Christopher J.

    2011-01-01

    Actin filament arrays are constantly remodeled as the needs of cells change as well as during responses to biotic and abiotic stimuli. Previous studies demonstrate that many single actin filaments in the cortical array of living Arabidopsis thaliana epidermal cells undergo stochastic dynamics, a combination of rapid growth balanced by disassembly from prolific severing activity. Filament turnover and dynamics are well understood from in vitro biochemical analyses and simple reconstituted systems. However, the identification in living cells of the molecular players involved in controlling actin dynamics awaits the use of model systems, especially ones where the power of genetics can be combined with imaging of individual actin filaments at high spatial and temporal resolution. Here, we test the hypothesis that actin depolymerizing factor (ADF)/cofilin contributes to stochastic filament severing and facilitates actin turnover. A knockout mutant for Arabidopsis ADF4 has longer hypocotyls and epidermal cells when compared with wild-type seedlings. This correlates with a change in actin filament architecture; cytoskeletal arrays in adf4 cells are significantly more bundled and less dense than in wild-type cells. Several parameters of single actin filament turnover are also altered. Notably, adf4 mutant cells have a 2.5-fold reduced severing frequency as well as significantly increased actin filament lengths and lifetimes. Thus, we provide evidence that ADF4 contributes to the stochastic dynamic turnover of actin filaments in plant cells. PMID:22010035

  5. The nuclear F-actin interactome of Xenopus oocytes reveals an actin-bundling kinesin that is essential for meiotic cytokinesis.

    PubMed

    Samwer, Matthias; Dehne, Heinz-Jürgen; Spira, Felix; Kollmar, Martin; Gerlich, Daniel W; Urlaub, Henning; Görlich, Dirk

    2013-07-01

    Nuclei of Xenopus laevis oocytes grow 100 000-fold larger in volume than a typical somatic nucleus and require an unusual intranuclear F-actin scaffold for mechanical stability. We now developed a method for mapping F-actin interactomes and identified a comprehensive set of F-actin binders from the oocyte nuclei. Unexpectedly, the most prominent interactor was a novel kinesin termed NabKin (Nuclear and meiotic actin-bundling Kinesin). NabKin not only binds microtubules but also F-actin structures, such as the intranuclear actin bundles in prophase and the contractile actomyosin ring during cytokinesis. The interaction between NabKin and F-actin is negatively regulated by Importin-β and is responsive to spatial information provided by RanGTP. Disconnecting NabKin from F-actin during meiosis caused cytokinesis failure and egg polyploidy. We also found actin-bundling activity in Nabkin's somatic paralogue KIF14, which was previously shown to be essential for somatic cell division. Our data are consistent with the notion that NabKin/KIF14 directly link microtubules with F-actin and that such link is essential for cytokinesis. PMID:23727888

  6. [The effect of Mg-ADP on the structural state of actin in the F-actin-myosin subfragment-1 complex].

    PubMed

    Borovikov, Iu S; Kirillina, V P

    1991-01-01

    Using polarized microfluorometry techniques, a study was made on the orientation and mobility of fluorescent probes 1,5-IAEDANS and rhomadin-phalloidin, located in various parts of actin, muscle fibers free of myosin, tropomyosin and troponin (ghost fibres) being used. It was found that the binding of a myosin subfragment 1 (S1) to actin induced changes in polarized fluorescence of the fibers. The analysis of these data showed that the formation of actin-S1 and actin-S1-ADP complexes in a muscle fiber resulted in a decrease in the angle between the thin filaments and the emission dipole of phalloidin-rhodamine, as well as in an increase of the mobility of this dye. In the experiments with the 1,5-IAEDANS label the angle of emission dipole increased, while the mobility of the label decreased. These changes were smaller in the presence of Mg-ADP than in its absence. It is assumed that the changes in actin monomer structure occur when a myosin head interacts with actin. These changes are expressed as those in orientation and mobility of large and small domains of actin in thin filaments. The domain orientation in actomyosin complex changes, influenced by Mg-ADP. The data obtained allow to propose the involvement of interdomain motions of some parts of actin monomer in the mechanisms of muscle contraction.

  7. Nuclear actin and protein 4.1: essential interactions during nuclear assembly in vitro.

    PubMed

    Krauss, Sharon Wald; Chen, Cynthia; Penman, Sheldon; Heald, Rebecca

    2003-09-16

    Structural protein 4.1, which has crucial interactions within the spectrin-actin lattice of the human red cell membrane skeleton, also is widely distributed at diverse intracellular sites in nucleated cells. We previously showed that 4.1 is essential for assembly of functional nuclei in vitro and that the capacity of 4.1 to bind actin is required. Here we report that 4.1 and actin colocalize in mammalian cell nuclei using fluorescence microscopy and, by higher-resolution detergent-extracted cell whole-mount electron microscopy, are associated on nuclear filaments. We also devised a cell-free assay using Xenopus egg extract containing fluorescent actin to follow actin during nuclear assembly. By directly imaging actin under nonperturbing conditions, the total nuclear actin population is retained and visualized in situ relative to intact chromatin. We detected actin initially when chromatin and nuclear pores began assembling. As nuclear lamina assembled, but preceding DNA synthesis, actin distributed in a reticulated pattern throughout the nucleus. Protein 4.1 epitopes also were detected when actin began to accumulate in nuclei, producing a diffuse coincident pattern. As nuclei matured, actin was detected both coincident with and also independent of 4.1 epitopes. To test whether acquisition of nuclear actin is required for nuclear assembly, the actin inhibitor latrunculin A was added to Xenopus egg extracts during nuclear assembly. Latrunculin A strongly perturbed nuclear assembly and produced distorted nuclear structures containing neither actin nor protein 4.1. Our results suggest that actin as well as 4.1 is necessary for nuclear assembly and that 4.1-actin interactions may be critical.

  8. Nuclear actin and protein 4.1: Essential interactions during nuclear assembly in vitro

    SciTech Connect

    Krauss, Sharon Wald; Chen, Cynthia; Penman, Sheldon; Heald, Rebecca

    2003-06-11

    Structural protein 4.1, which has crucial interactions within the spectin-actin lattice of the human red cell membrane skeleton, also is widely distributed at diverse intracellular sites in nucleated cells. We previously showed that 4.1 is essential for assembly of functional nuclei in vitro and that the capacity of 4.1 to bind actin is required. Here we report that 4.1 and actin colocalize in mammalian cell nuclei using fluorescence microscopy and, by higher resolution cell whole mount electron microscopy, are associated on nuclear filaments. We also devised a cell-free assay using Xenopus egg extract containing fluorescent actin to follow actin during nuclear assembly. By directly imaging actin under non-perturbing conditions, the total nuclear actin population is retained and is visualized in situ relative to intact chromatin. We detected actin initially when chromatin and nuclear pores began assembling. As the nuclear lamina assembled, but preceding DNA synthesis, a discrete actin network formed throughout the nucleus. Protein 4.1 epitopes also were detected when actin began to accumulate in nuclei, producing a diffuse coincident pattern. As nuclei matured, actin was detected both coincident with and also independent of 4.1 epitopes. To test whether acquisition of nuclear actin is required for nuclear assembly, the actin inhibitor latrunculin A was added to Xenopus egg extracts during nuclear assembly. Latrunculin A strongly perturbed nuclear assembly and produced distorted nuclear structures containing neither actin nor protein 4.1. Our results suggest that actin as well as 4.1 is necessary for nuclear assembly and that 4.1-actin interactions may be critical.

  9. Actin-Dependent Alterations of Dendritic Spine Morphology in Shankopathies

    PubMed Central

    Sarowar, Tasnuva

    2016-01-01

    Shank proteins (Shank1, Shank2, and Shank3) act as scaffolding molecules in the postsynaptic density of many excitatory neurons. Mutations in SHANK genes, in particular SHANK2 and SHANK3, lead to autism spectrum disorders (ASD) in both human and mouse models. Shank3 proteins are made of several domains—the Shank/ProSAP N-terminal (SPN) domain, ankyrin repeats, SH3 domain, PDZ domain, a proline-rich region, and the sterile alpha motif (SAM) domain. Via various binding partners of these domains, Shank3 is able to bind and interact with a wide range of proteins including modulators of small GTPases such as RICH2, a RhoGAP protein, and βPIX, a RhoGEF protein for Rac1 and Cdc42, actin binding proteins and actin modulators. Dysregulation of all isoforms of Shank proteins, but especially Shank3, leads to alterations in spine morphogenesis, shape, and activity of the synapse via altering actin dynamics. Therefore, here, we highlight the role of Shank proteins as modulators of small GTPases and, ultimately, actin dynamics, as found in multiple in vitro and in vivo models. The failure to mediate this regulatory role might present a shared mechanism in the pathophysiology of autism-associated mutations, which leads to dysregulation of spine morphogenesis and synaptic signaling. PMID:27795858

  10. Rapid actin-dependent viral motility in live cells.

    PubMed

    Vaughan, Joshua C; Brandenburg, Boerries; Hogle, James M; Zhuang, Xiaowei

    2009-09-16

    During the course of an infection, viruses take advantage of a variety of mechanisms to travel in cells, ranging from diffusion within the cytosol to active transport along cytoskeletal filaments. To study viral motility within the intrinsically heterogeneous environment of the cell, we have developed a motility assay that allows for the global and unbiased analysis of tens of thousands of virus trajectories in live cells. Using this assay, we discovered that poliovirus exhibits anomalously rapid intracellular movement that was independent of microtubules, a common track for fast and directed cargo transport. Such rapid motion, with speeds of up to 5 microm/s, allows the virus particles to quickly explore all regions of the cell with the exception of the nucleus. The rapid, microtubule-independent movement of poliovirus was observed in multiple human-derived cell lines, but appeared to be cargo-specific. Other cargo, including a closely related picornavirus, did not exhibit similar motility. Furthermore, the motility is energy-dependent and requires an intact actin cytoskeleton, suggesting an active transport mechanism. The speed of this microtubule-independent but actin-dependent movement is nearly an order of magnitude faster than the fastest speeds reported for actin-dependent transport in animal cells, either by actin polymerization or by myosin motor proteins.

  11. Role of gelsolin in actin depolymerization of adherent human neutrophils.

    PubMed Central

    Wang, J S; Coburn, J P; Tauber, A I; Zaner, K S

    1997-01-01

    Human neutrophils generally function adherent to an extracellular matrix. We have previously reported that upon adhesion to laminin- or fibronectin-coated, but not uncoated, plastic there is a depolymerization of actin in neutrophils. This phenomenon was not affected by inhibitors of the more well-studied components of the signal transduction pathway, specifically, pertussis toxin, an inhibitor of G-proteins, H-7 or staurosporine, inhibitors of protein kinase C, or herbimycin A, an inhibitor of nonreceptor tyrosine kinase. We therefore focused our attention on actin-binding proteins and measured the changes in the partitioning of gelsolin between the Triton X-100-soluble and -insoluble cellular fractions which occur upon neutrophil adhesion by means of quantitating anti-gelsolin antibody binding to aliquots of these fractions. It was found that approximately 90% of the total cellular gelsolin was found in the Triton X-100-soluble fraction in suspended cells, but that upon adherence to either fibronectin- or laminin-coated plastic about 40% of the soluble gelsolin could be detected in the insoluble fraction. This effect was not observed in cells adherent to uncoated plastic, wherein more than 90% of the gelsolin was found in the soluble fraction. Results of immunofluorescence microscopy of these cell preparations was consistent with this data. A gelsolin translocation to the insoluble cellular actin network may account for a part of the observed actin depolymerization. Images PMID:9017600

  12. Supervillin Reorganizes the Actin Cytoskeleton and Increases Invadopodial Efficiency

    PubMed Central

    Crowley, Jessica L.; Smith, Tara C.; Fang, Zhiyou; Takizawa, Norio

    2009-01-01

    Tumor cells use actin-rich protrusions called invadopodia to degrade extracellular matrix (ECM) and invade tissues; related structures, termed podosomes, are sites of dynamic ECM interaction. We show here that supervillin (SV), a peripheral membrane protein that binds F-actin and myosin II, reorganizes the actin cytoskeleton and potentiates invadopodial function. Overexpressed SV induces redistribution of lamellipodial cortactin and lamellipodin/RAPH1/PREL1 away from the cell periphery to internal sites and concomitantly increases the numbers of F-actin punctae. Most punctae are highly dynamic and colocalize with the podosome/invadopodial proteins, cortactin, Tks5, and cdc42. Cortactin binds SV sequences in vitro and contributes to the formation of enhanced green fluorescent protein (EGFP)-SV induced punctae. SV localizes to the cores of Src-generated podosomes in COS-7 cells and with invadopodia in MDA-MB-231 cells. EGFP-SV overexpression increases average numbers of ECM holes per cell; RNA interference-mediated knockdown of SV decreases these numbers. Although SV knockdown alone has no effect, simultaneous down-regulation of SV and the closely related protein gelsolin reduces invasion through ECM. Together, our results show that SV is a component of podosomes and invadopodia and that SV plays a role in invadopodial function, perhaps as a mediator of cortactin localization, activation state, and/or dynamics of metalloproteinases at the ventral cell surface. PMID:19109420

  13. A myopathy-related actin mutation increases contractile function.

    PubMed

    Lindqvist, Johan; Pénisson-Besnier, Isabelle; Iwamoto, Hiroyuki; Li, Meishan; Yagi, Naoto; Ochala, Julien

    2012-05-01

    Nemaline myopathy (NM) is the most common congenital myopathy and is caused by mutations in various genes including NEB (nebulin), TPM2 (beta-tropomyosin), TPM3 (gamma-tropomyosin), and ACTA1 (skeletal alpha-actin). 20-25% of NM cases carry ACTA1 defects and these particular mutations usually induce substitutions of single residues in the actin protein. Despite increasing clinical and scientific interest, the contractile consequences of these subtle amino acid substitutions remain obscure. To decipher them, in the present study, we originally recorded and analysed the mechanics as well as the X-ray diffraction patterns of human membrane-permeabilized single muscle fibres with a particular peptide substitution in actin, i.e. p.Phe352Ser. Results unravelled an unexpected cascade of molecular and cellular events. During contraction, p.Phe352Ser greatly enhances the strain of individual cross-bridges. Paradoxically, p.Phe352Ser also slightly lowers the number of cross-bridges by altering the rate of myosin head attachment to actin monomers. Overall, at the cell level, these divergent mechanisms conduct to an improved steady-state force production. Such results provide new surprising scientific insights and crucial information for future therapeutic strategies. PMID:22358459

  14. Severe congenital actin related myopathy with myofibrillar myopathy features.

    PubMed

    Selcen, Duygu

    2015-06-01

    Mutations in ACTA1 have been associated with different pathologic findings including nemaline myopathy, intranuclear rod myopathy, actin myopathy, cap myopathy, congenital fiber type disproportion, and core myopathy. Myofibrillar myopathies are morphologically distinct but genetically heterogeneous muscular dystrophies arising from mutations in Z-disk related proteins. We report a 26-month-old boy with significantly delayed motor development requiring mechanical ventilation and tube-feeding since birth. The muscle biopsy displayed typical features of myofibrillar myopathy with abnormal expression of multiple proteins. Whole exome sequencing revealed two-amino-acid duplication in ACTA1. In cell culture system, mutant actin was expressed at ~11% of wild-type, and mutant actin formed pleomorphic cytoplasmic aggregates whereas wild-type actin appeared in filamentous structures. We conclude that mutations in ACTA1 can cause pathologic features consistent with myofibrillar myopathy, and mutations in ACTA1 should be considered in patients with severe congenital hypotonia associated with muscle weakness and features of myofibrillar myopathy. PMID:25913210

  15. Mechanics of F-actin characterized with microfabricated cantilevers.

    PubMed

    Liu, Xiumei; Pollack, Gerald H

    2002-11-01

    In this report we characterized the longitudinal elasticity of single actin filaments manipulated by novel silicon-nitride microfabricated levers. Single actin filaments were stretched from zero tension to maximal physiological tension, P(0). The obtained length-tension relation was nonlinear in the low-tension range (0-50 pN) with a resultant strain of approximately 0.4-0.6% and then became linear at moderate to high tensions (approximately 50-230 pN). In this region, the stretching stiffness of a single rhodamine-phalloidin-labeled, 1-microm-long F-actin is 34.5 +/- 3.5 pN/nm. Such a length-tension relation could be characterized by an entropic-enthalpic worm-like chain model, which ascribes most of the energy consumed in the nonlinear portion to overcoming thermal undulations arising from the filament's interaction with surrounding solution and the linear portion to the intrinsic stretching elasticity. By fitting the experimental data with such a worm-like chain model, an estimation of persistence length of approximately 8.75 microm was derived. These results suggest that F-actin is more compliant than previously thought and that thin filament compliance may account for a substantial fraction of the sarcomere's elasticity.

  16. The Actin Cortex: A Bridge between Cell Shape and Function.

    PubMed

    Chalut, Kevin J; Paluch, Ewa K

    2016-09-26

    The cortical actin network controls many animal cell shape changes by locally modulating cortical tension. Recent work has provided insight into cortex components and regulators. However, how the network is reorganized in response to cellular signaling, and the role reorganization may play during cell state changes, remain to be determined. PMID:27676427

  17. Actin filament dynamics impacts keratinocyte stem cell maintenance

    PubMed Central

    Nanba, Daisuke; Toki, Fujio; Matsushita, Natsuki; Matsushita, Sachi; Higashiyama, Shigeki; Barrandon, Yann

    2013-01-01

    Cultured human epidermal keratinocyte stem cells (holoclones) are crucial for regenerative medicine for burns and genetic disorders. In serial culture, holoclones progressively lose their proliferative capacity to become transient amplifying cells with limited growth (paraclones), a phenomenon termed clonal conversion. Although it negatively impacts the culture lifespan and the success of cell transplantation, little is known on the molecular mechanism underlying clonal conversion. Here, we show that holoclones and paraclones differ in their actin filament organization, with actin bundles distributed radially in holoclones and circumferentially in paraclones. Moreover, actin organization sets the stage for a differing response to epidermal growth factor (EGF), since EGF signalling induces a rapid expansion of colony size in holoclones and a significant reduction in paraclones. Furthermore, inhibition of PI3K or Rac1 in holoclones results in the reorganization of actin filaments in a pattern that is similar to that of paraclones. Importantly, continuous Rac1 inhibition in holoclones results in clonal conversion and reduction of growth potential. Together, our data connect loss of stem cells to EGF-induced colony dynamics governed by Rac1. PMID:23554171

  18. Comparison Actin- and Glass-Supported Phospholipid Bilayer Diffusion Coefficients

    PubMed Central

    Sterling, Sarah M.; Dawes, Ryan; Allgeyer, Edward S.; Ashworth, Sharon L.; Neivandt, David J.

    2015-01-01

    The formation of biomimetic lipid membranes has the potential to provide insights into cellular lipid membrane dynamics. The construction of such membranes necessitates not only the utilization of appropriate lipids, but also physiologically relevant substrate/support materials. The substrate materials employed have been shown to have demonstrable effects on the behavior of the overlying lipid membrane, and thus must be studied before use as a model cushion support. To our knowledge, we report the formation and investigation of a novel actin protein-supported lipid membrane. Specifically, inner leaflet lateral mobility of globular actin-supported DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) bilayers, deposited via the Langmuir-Blodgett/Langmuir Schaefer methodology, was investigated by z-scan fluorescence correlation spectroscopy across a temperature range of 20–44°C. The actin substrate was found to decrease the diffusion coefficient when compared to an identical membrane supported on glass. The depression of the diffusion coefficient occurred across all measured temperatures. These results indicated that the actin substrate exerted a direct effect on the fluidity of the lipid membrane and highlighted the fact that the choice of substrate/support is critical in studies of model lipid membranes. PMID:25902434

  19. Evaluation of actinic cheilitis using fluorescence lifetime spectroscopy

    NASA Astrophysics Data System (ADS)

    Saito Nogueira, Marcelo; Cosci, Alessandro; Pratavieira, Sebastião.; Takahama, Ademar; Souza Azevedo, Rebeca; Kurachi, Cristina

    2016-03-01

    Actinic cheilitis is a potentially malignant disorder that mostly affects the vermilion border of the lower lip and can lead to squamous cell carcinoma. Because of its heterogeneous clinical aspect, it is difficult to indicate representative biopsy area. Late diagnosis is a limiting factor of therapeutic possibilities available to treat oral cancer. The diagnosis of actinic cheilitis is mainly based on clinical and histopathological analysis and it is a time consuming procedure to get the results. Information about the organization and chemical composition of the tissues can be obtained using fluorescence lifetime spectroscopy techniques without the need for biopsy. The main targeted fluorophores are NADH (nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide), which have free and bound states, each one with different average lifetimes. The average lifetimes for free and bound NADH and FAD change according to tissue metabolic alterations and allow a quick and non-invasive clinical investigation of injuries and to help clinicians with the early diagnosis of actinic cheilitis. This study aims to evaluate the fluorescence lifetime parameters at the discrimination of three degrees of epithelial dysplasia, the most important predictor of malignant development, described in up to 100% of actinic cheilitis cases.

  20. Calcium Regulation Of Actin Filament Speed In Vitro

    NASA Astrophysics Data System (ADS)

    Lamadrid, M. A.; Gordon, A. M.; Chase, P. B.; Chen, Y.; Luo, Z.

    1998-03-01

    Using an in-vitro motility assay, we have studied the Ca regulation of the gliding speed of actin filaments with regulatory proteins troponin and tropomyosin. In skeletal muscle, Ca binding to the troponin/tropomyosin system serves as the switch which enables a myosin head to bind to actin and create a power stroke. Fluorescently labeled filaments were observed using video fluorescence microscopy and speeds measured for different calcium concentrations and ionic strengths. In contrast to F-actin (for which speed was unaffected by [Ca]), the speed increased with increasing [Ca] in a non-linear manner. By comparing the behavior of short and long filaments, we also found that there was no length dependence to the observed non-zero speeds, but filaments shorter than 3 um had a higher tendency to undergo stop-go motion. Analysis of the data in the context of protein friction suggests that Ca affects not only the number of binding motors but also the lifetime of the strong binding state between actin and myosin.