Defects in G-Actin Incorporation into Filaments in Myoblasts Derived from Dysferlinopathy Patients Are Restored by Dysferlin C2 Domains

Dysferlin is a transmembrane C-2 domain-containing protein involved in vesicle trafficking and membrane remodeling in skeletal muscle cells. However, the mechanism by which dysferlin regulates these cellular processes remains unclear. Since actin dynamics is critical for vesicle trafficking and membrane remodeling, we studied the role of dysferlin in Ca2+-induced G-actin incorporation into filaments in four different immortalized myoblast cell lines (DYSF2, DYSF3, AB320, and ER) derived from patients harboring mutations in the dysferlin gene. As compared with immortalized myoblasts obtained from a control subject, dysferlin expression and G-actin incorporation were significantly decreased in myoblasts from dysferlinopathy patients. Stable knockdown of dysferlin with specific shRNA in control myoblasts also significantly reduced G-actin incorporation. The impaired G-actin incorporation was restored by the expression of full-length dysferlin as well as dysferlin N-terminal or C-terminal regions, both of which contain three C2 domains. DYSF3 myoblasts also exhibited altered distribution of annexin A2, a dysferlin partner involved in actin remodeling. However, dysferlin N-terminal and C-terminal regions appeared to not fully restore such annexin A2 mislocation. Then, our results suggest that dysferlin regulates actin remodeling by a mechanism that does to not involve annexin A2.

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The Role of BAR Domain Proteins in the Regulation of Membrane Dynamics

Acta Naturae ◽

10.32607/20758251-2016-8-4-60-69 ◽

2016 ◽

Vol 8 (4) ◽

pp. 60-69 ◽

Cited By ~ 18

Author(s):

T. B. Stanishneva-Konovalova ◽

N. I Derkacheva ◽

S. V. Polevova ◽

O. S. Sokolova

Keyword(s):

Membrane Dynamics ◽

Actin Dynamics ◽

Membrane Remodeling ◽

Bar Domain ◽

Cellular Processes ◽

Functional Differences ◽

Bar Domain Proteins ◽

Domain Protein ◽

Bar Domains

Many cellular processes are associated with membrane remodeling. The BAR domain protein family plays a key role in the formation and detection of local membrane curvatures and in attracting other proteins, including the regulators of actin dynamics. Based on their structural and phylogenetic properties, BAR domains are divided into several groups which affect membrane in various ways and perform different functions in cells. However, recent studies have uncovered evidence of functional differences even within the same group. This review discusses the principles underlying the interactions of different groups of BAR domains, and their individual representatives, with membranes.

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Dynein Light Chain 1 Regulates Dynamin-mediated F-Actin Assembly during Sperm Individualization in Drosophila

Molecular Biology of the Cell ◽

10.1091/mbc.e05-02-0103 ◽

2005 ◽

Vol 16 (7) ◽

pp. 3107-3116 ◽

Cited By ~ 29

Author(s):

Anindya Ghosh-Roy ◽

Bela S. Desai ◽

Krishanu Ray

Keyword(s):

Light Chain ◽

Cytoplasmic Dynein ◽

Actin Dynamics ◽

Dynein Light Chain ◽

Actin Assembly ◽

Cellular Functions ◽

Directed Movement ◽

Cellular Processes ◽

Dynactin Complex

Toward the end of spermiogenesis, spermatid nuclei are compacted and the clonally related spermatids individualize to become mature and active sperm. Studies in Drosophila showed that caudal end-directed movement of a microfilament-rich structure, called investment cone, expels the cytoplasmic contents of individual spermatids. F-actin dynamics plays an important role in this process. Here we report that the dynein light chain 1 (DLC1) of Drosophila is involved in two separate cellular processes during sperm individualization. It is enriched around spermatid nuclei during postelongation stages and plays an important role in the dynein-dynactin–dependent rostral retention of the nuclei during this period. In addition, DDLC1 colocalizes with dynamin along investment cones and regulates F-actin assembly at this organelle by retaining dynamin along the cones. Interestingly, we found that this process does not require the other subunits of cytoplasmic dynein-dynactin complex. Altogether, these observations suggest that DLC1 could independently regulate multiple cellular functions and established a novel role of this protein in F-actin assembly in Drosophila.

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Withaferin A binds covalently to the N-terminal domain of annexin A2

Biological Chemistry ◽

10.1515/hsz-2012-0184 ◽

2012 ◽

Vol 393 (10) ◽

pp. 1151-1163 ◽

Cited By ~ 17

Author(s):

Gabriel Ozorowski ◽

Christopher M. Ryan ◽

Julian P. Whitelegge ◽

Hartmut Luecke

Keyword(s):

Actin Polymerization ◽

Inhibitory Effect ◽

Annexin A2 ◽

Actin Dynamics ◽

Withaferin A ◽

Core Domain ◽

Cancer Pathways ◽

Terminal Domain ◽

C Terminus

Abstract Annexin A2 (AnxA2), a 38-kDa member of the Ca2+-binding annexin family, has been implicated in numerous cancer pathways. Withaferin A (WithfA), a natural plant compound, has been reported previously to bind covalently to Cys133 of the AnxA2 core domain leading to a reduction of the invasive capabilities of cancer cells by altering their cytoskeleton. We show here that AnxA2 has an inhibitory effect on actin polymerization, and a modification with WithfA significantly increases this inhibitory role of AnxA2. Using mass spectrometry and single-site mutants, we localized the WithfA-AnxA2 interaction to the N-terminal domain of AnxA2 where WithfA binds covalently to Cys9. Whereas binding to F-actin filaments has been mapped to the C terminus of AnxA2, our results suggest that the N-terminal domain modified by WithfA may also play a role in the AnxA2-actin interaction. The binding of WithfA may regulate the AnxA2-mediated actin dynamics in two distinct ways: (i) the increase of F-actin bundling activity by the Anx2/p11 heterotetramer and (ii) the decrease of actin polymerization as a result of the increased affinity of AnxA2 to the barbed end of actin microfilaments. We demonstrate the susceptibility of Cys9 of AnxA2 to chemical modifications and exclude Cys133 as a binding site for WithfA.

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Muscling in on mitochondrial sexual dimorphism; role of mitochondrial dimorphism in skeletal muscle health and disease

Clinical Science ◽

10.1042/cs20160940 ◽

2017 ◽

Vol 131 (15) ◽

pp. 1919-1922 ◽

Cited By ~ 3

Author(s):

Gareth A. Nye ◽

Giorgos K. Sakellariou ◽

Hans Degens ◽

Adam P. Lightfoot

Keyword(s):

Skeletal Muscle ◽

Sexual Dimorphism ◽

Mitochondrial Function ◽

FMRP Modulates Activity-Dependent Spine Plasticity by Binding Cofilin1 mRNA and Regulating Localization and Local Translation

Cerebral Cortex ◽

10.1093/cercor/bhz059 ◽

2019 ◽

Vol 29 (12) ◽

pp. 5204-5216 ◽

Cited By ~ 4

Author(s):

Jonas Feuge ◽

Franziska Scharkowski ◽

Kristin Michaelsen-Preusse ◽

Martin Korte

Keyword(s):

Fragile X Syndrome ◽

Fragile X ◽

Treatment Strategies ◽

Actin Dynamics ◽

Actin Remodeling ◽

Dendritic Spine Morphology ◽

Novel Treatment Strategies ◽

Activity Dependent ◽

Spine Plasticity

Abstract Multiple variants of intellectual disability, e.g., the Fragile X Syndrome are associated with alterations in dendritic spine morphology, thereby pointing to dysregulated actin dynamics during development and processes of synaptic plasticity. Surprisingly, although the necessity of spine actin remodeling was demonstrated repeatedly, the importance and precise role of actin regulators is often undervalued. Here, we provide evidence that structural and functional plasticity are severely impaired after NMDAR-dependent LTP in the hippocampus of Fmr1 KO mice. We can link these defects to an aberrant activity-dependent regulation of Cofilin 1 (cof1) as activity-dependent modulations of local cof1 mRNA availability, local cof1 translation as well as total cof1 expression are impaired in the absence of FMRP. Finally, we can rescue activity-dependent structural plasticity in KO neurons by mimicking the regulation of cof1 observed in WT cells, thereby illustrating the potential of actin modulators to provide novel treatment strategies for the Fragile X Syndrome.

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The PDZ-adaptor protein syntenin-1 regulates HIV-1 entry

Molecular Biology of the Cell ◽

10.1091/mbc.e11-12-1003 ◽

2012 ◽

Vol 23 (12) ◽

pp. 2253-2263 ◽

Cited By ~ 22

Author(s):

Mónica Gordón-Alonso ◽

Vera Rocha-Perugini ◽

Susana Álvarez ◽

Olga Moreno-Gonzalo ◽

Ángeles Ursa ◽

...

Keyword(s):

Actin Polymerization ◽

Adaptor Protein ◽

Viral Receptor ◽

Actin Remodeling ◽

Cellular Processes ◽

Immunodeficiency Virus ◽

Viral Nucleocapsid ◽

Hiv 1

Syntenin-1 is a cytosolic adaptor protein involved in several cellular processes requiring polarization. Human immunodeficiency virus type 1 (HIV-1) attachment to target CD4+T-cells induces polarization of the viral receptor and coreceptor, CD4/CXCR4, and cellular structures toward the virus contact area, and triggers local actin polymerization and phosphatidylinositol 4,5-bisphosphate (PIP2) production, which are needed for successful HIV infection. We show that syntenin-1 is recruited to the plasma membrane during HIV-1 attachment and associates with CD4, the main HIV-1 receptor. Syntenin-1 overexpression inhibits HIV-1 production and HIV-mediated cell fusion, while syntenin depletion specifically increases HIV-1 entry. Down-regulation of syntenin-1 expression reduces F-actin polymerization in response to HIV-1. Moreover, HIV-induced PIP2accumulation is increased in syntenin-1–depleted cells. Once the virus has entered the target cell, syntenin-1 polarization toward the viral nucleocapsid is lost, suggesting a spatiotemporal regulatory role of syntenin-1 in actin remodeling, PIP2production, and the dynamics of HIV-1 entry.

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A pharmacological cocktail for arresting actin dynamics in living cells

Molecular Biology of the Cell ◽

10.1091/mbc.e11-04-0379 ◽

2011 ◽

Vol 22 (21) ◽

pp. 3986-3994 ◽

Cited By ~ 53

Author(s):

Grace E. Peng ◽

Sarah R. Wilson ◽

Orion D. Weiner

Keyword(s):

Leading Edge ◽

Actin Dynamics ◽

Actin Assembly ◽

Actin Reorganization ◽

Cellular Processes ◽

Wide Range ◽

Morphological Polarity ◽

Nih 3T3 ◽

External Stimuli

The actin cytoskeleton is regulated by factors that influence polymer assembly, disassembly, and network rearrangement. Drugs that inhibit these events have been used to test the role of actin dynamics in a wide range of cellular processes. Previous methods of arresting actin rearrangements take minutes to act and work well in some contexts, but can lead to significant actin reorganization in cells with rapid actin dynamics, such as neutrophils. In this paper, we report a pharmacological cocktail that not only arrests actin dynamics but also preserves the structure of the existing actin network in neutrophil-like HL-60 cells, human fibrosarcoma HT1080 cells, and mouse NIH 3T3 fibroblast cells. Our cocktail induces an arrest of actin dynamics that initiates within seconds and persists for longer than 10 min, during which time cells maintain their responsivity to external stimuli. With this cocktail, we demonstrate that actin dynamics, and not simply morphological polarity or actin accumulation at the leading edge, are required for the spatial persistence of Rac activation in HL-60 cells. Our drug combination preserves the structure of the existing cytoskeleton while blocking actin assembly, disassembly, and rearrangement, and should prove useful for investigating the role of actin dynamics in a wide range of cellular signaling contexts.

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p21-Activated kinases regulate actin remodeling in glomerular podocytes

AJP Renal Physiology ◽

10.1152/ajprenal.00536.2009 ◽

2010 ◽

Vol 298 (4) ◽

pp. F951-F961 ◽

Cited By ~ 19

Author(s):

Jianxin Zhu ◽

Ortal Attias ◽

Lamine Aoudjit ◽

Ruihua Jiang ◽

Hiroshi Kawachi ◽

...

Keyword(s):

Actin Cytoskeleton ◽

Tyrosine Phosphorylation ◽

Adaptor Proteins ◽

Dominant Negative ◽

Puromycin Aminonucleoside ◽

Actin Remodeling ◽

Cellular Processes ◽

Aminonucleoside Nephrosis ◽

Puromycin Aminonucleoside Nephrosis

The tyrosine phosphorylation of nephrin is reported to regulate podocyte morphology via the Nck adaptor proteins. The Pak family of kinases are regulators of the actin cytoskeleton and are recruited to the plasma membrane via Nck. Here, we investigated the role of Pak in podocyte morphology. Pak1/2 were expressed in cultured podocytes. In mouse podocytes, Pak2 was predominantly phosphorylated, concentrated at the tips of the cellular processes, and its expression and/or phosphorylation were further increased when differentiated. Overexpression of rat nephrin in podocytes increased Pak1/2 phosphorylation, which was abolished when the Nck binding sites were mutated. Furthermore, dominant-negative Nck constructs blocked the Pak1 phosphorylation induced by antibody-mediated cross linking of nephrin. Transient transfection of constitutively kinase-active Pak1 into differentiated mouse podocytes decreased stress fibers, increased cortical F-actin, and extended the cellular processes, whereas kinase-dead mutant, kinase inhibitory construct, and Pak2 knockdown by shRNA had the opposite effect. In a rat model of puromycin aminonucleoside nephrosis, Pak1/2 phosphorylation was decreased in glomeruli, concomitantly with a decrease of nephrin tyrosine phosphorylation. These results suggest that Pak contributes to remodeling of the actin cytoskeleton in podocytes. Disturbed nephrin-Nck-Pak interaction may contribute to abnormal morphology of podocytes and proteinuria.

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The noncanonical role of the protease cathepsin D as a cofilin phosphatase

Cell Research ◽

10.1038/s41422-020-00454-w ◽

2021 ◽

Author(s):

Yi-Jun Liu ◽

Ting Zhang ◽

Sicong Chen ◽

Daxiao Cheng ◽

Cunjin Wu ◽

...

Keyword(s):

Cathepsin D ◽

Dual Function ◽

Actin Remodeling ◽

Actin Organization ◽

Cellular Processes ◽

Lysosomal Protease ◽

Ph Conditions ◽

Maturation State ◽

Acidic Compartments

AbstractCathepsin D (cathD) is traditionally regarded as a lysosomal protease that degrades substrates in acidic compartments. Here we report cathD plays an unconventional role as a cofilin phosphatase orchestrating actin remodeling. In neutral pH environments, the cathD precursor directly dephosphorylates and activates the actin-severing protein cofilin independent of its proteolytic activity, whereas mature cathD degrades cofilin in acidic pH conditions. During development, cathD complements the canonical cofilin phosphatase slingshot and regulates the morphogenesis of actin-based structures. Moreover, suppression of cathD phosphatase activity leads to defective actin organization and cytokinesis failure. Our findings identify cathD as a dual-function molecule, whose functional switch is regulated by environmental pH and its maturation state, and reveal a novel regulatory role of cathD in actin-based cellular processes.

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Differential physiological role of BIN1 isoforms in skeletal muscle development, function and regeneration

10.1101/477950 ◽

2018 ◽

Author(s):

Ivana Prokic ◽

Belinda Cowling ◽

Candice Kutchukian ◽

Christine Kretz ◽

Hichem Tasfaout ◽

...

Keyword(s):

Skeletal Muscle ◽

Muscle Regeneration ◽

Muscle Development ◽

Physiological Role ◽

Membrane Remodeling ◽

Skeletal Muscle Development ◽

Developmental Defects ◽

Intracellular Organization ◽

And Function

AbstractSkeletal muscle development and regeneration are tightly regulated processes. How the intracellular organization of muscle fibers is achieved during these steps is unclear. Here we focus on the cellular and physiological roles of amphiphysin 2 (BIN1), a membrane remodeling protein mutated in both congenital and adult centronuclear myopathies, that is ubiquitously expressed and has skeletal muscle-specific isoforms. We created and characterized constitutive, muscle-specific and inducible Bin1 homozygous and heterozygous knockout mice targeting either ubiquitous or muscle-specific isoforms. Constitutive Bin1-deficient mice died at birth from lack of feeding due to a skeletal muscle defect. T-tubules and other organelles were misplaced and altered, supporting a general early role of BIN1 on intracellular organization in addition to membrane remodeling. Whereas restricted deletion of Bin1 in unchallenged adult muscles had no impact, the forced switch from the muscle-specific isoforms to the ubiquitous isoforms through deletion of the in-frame muscle–specific exon delayed muscle regeneration. Thus, BIN1 ubiquitous function is necessary for muscle development and function while its muscle-specific isoforms fine-tune muscle regeneration in adulthood, supporting that BIN1 centronuclear myopathy with congenital onset are due to developmental defects while later onset may be due to regeneration defects.

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