scholarly journals Cyclase-associated Protein 1 (CAP1) Promotes Cofilin-induced Actin Dynamics in Mammalian Nonmuscle Cells

2004 ◽  
Vol 15 (5) ◽  
pp. 2324-2334 ◽  
Author(s):  
Enni Bertling ◽  
Pirta Hotulainen ◽  
Pieta K. Mattila ◽  
Tanja Matilainen ◽  
Marjo Salminen ◽  
...  
Keyword(s):  
Actin Dynamics ◽  
Striated Muscles ◽  
Actin Monomer ◽  
Cortical Actin ◽  
Cellular Processes ◽  
Nonmuscle Cells ◽  
Associated Proteins ◽  
Dynamic Regions ◽  
And Function

Cyclase-associated proteins (CAPs) are highly conserved actin monomer binding proteins present in all eukaryotes. However, the mechanism by which CAPs contribute to actin dynamics has been elusive. In mammals, the situation is further complicated by the presence of two CAP isoforms whose differences have not been characterized. Here, we show that CAP1 is widely expressed in mouse nonmuscle cells, whereas CAP2 is the predominant isoform in developing striated muscles. In cultured NIH3T3 and B16F1 cells, CAP1 is a highly abundant protein that colocalizes with cofilin-1 to dynamic regions of the cortical actin cytoskeleton. Analysis of CAP1 knockdown cells demonstrated that this protein promotes rapid actin filament depolymerization and is important for cell morphology, migration, and endocytosis. Interestingly, depletion of CAP1 leads to an accumulation of cofilin-1 into abnormal cytoplasmic aggregates and to similar cytoskeletal defects to those seen in cofilin-1 knockdown cells, demonstrating that CAP1 is required for proper subcellular localization and function of ADF/cofilin. Together, these data provide the first direct in vivo evidence that CAP promotes rapid actin dynamics in conjunction with ADF/cofilin and is required for several central cellular processes in mammals.

scholarly journals In Vivo Role of Focal Adhesion Kinase in Regulating Pancreatic  -Cell Mass and Function Through Insulin Signaling, Actin Dynamics, and Granule Trafficking

Diabetes ◽  
2012 ◽  
Vol 61 (7) ◽  
pp. 1708-1718 ◽  
Author(s):  
E. P. Cai ◽  
M. Casimir ◽  
S. A. Schroer ◽  
C. T. Luk ◽  
S. Y. Shi ◽  
...  
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Insulin Signaling ◽  
Cell Mass ◽  
Actin Dynamics ◽  
Pancreatic Cell ◽  
And Function

scholarly journals Dynamic Localization and Function of Bni1p at the Sites of Directed Growth in Saccharomyces cerevisiae

2001 ◽  
Vol 21 (3) ◽  
pp. 827-839 ◽  
Author(s):  
Kumi Ozaki-Kuroda ◽  
Yasunori Yamamoto ◽  
Hidenori Nohara ◽  
Makoto Kinoshita ◽  
Takeshi Fujiwara ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fluorescent Protein ◽  
Imaging System ◽  
Cell Polarization ◽  
Actin Binding ◽  
Cortical Actin ◽  
Dynamic Localization ◽  
Directed Growth ◽  
And Function

ABSTRACT Formin homology (FH) proteins are implicated in cell polarization and cytokinesis through actin organization. There are two FH proteins in the yeast Saccharomyces cerevisiae, Bni1p and Bnr1p. Bni1p physically interacts with Rho family small G proteins (Rho1p and Cdc42p), actin, two actin-binding proteins (profilin and Bud6p), and a polarity protein (Spa2p). Here we analyzed the in vivo localization of Bni1p by using a time-lapse imaging system and investigated the regulatory mechanisms of Bni1p localization and function in relation to these interacting proteins. Bni1p fused with green fluorescent protein localized to the sites of cell growth throughout the cell cycle. In a small-budded cell, Bni1p moved along the bud cortex. This dynamic localization of Bni1p coincided with the apparent site of bud growth. Abni1-disrupted cell showed a defect in directed growth to the pre-bud site and to the bud tip (apical growth), causing its abnormally spherical cell shape and thick bud neck. Bni1p localization at the bud tips was absolutely dependent on Cdc42p, largely dependent on Spa2p and actin filaments, and partly dependent on Bud6p, but scarcely dependent on polarized cortical actin patches or Rho1p. These results indicate that Bni1p regulates polarized growth within the bud through its unique and dynamic pattern of localization, dependent on multiple factors, including Cdc42p, Spa2p, Bud6p, and the actin cytoskeleton.

Small-Molecule Screen Identifies ROS as Key Regulators of Actin Dynamics in Neutrophils

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4641-4641
Author(s):  
Hidenori Hattori ◽  
Kulandayan K Subramanian ◽  
Hongbo R. Luo
Keyword(s):  
Small Molecule ◽  
Actin Polymerization ◽  
Neutrophil Migration ◽  
Physiological Role ◽  
Leading Edge ◽  
Actin Dynamics ◽  
Functional Screening ◽  
Cellular Processes

Abstract Precise spatial and temporal control of actin polymerization and depolymerization is essential for mediating various cellular processes such as migration, phagocytosis, vesicle trafficking and adhesion. In this study, we used a small-molecule functional screening approach to identify novel regulators of actin dynamics during neutrophil migration. Here we show that NADPH-oxidase dependent Reactive Oxygen Species act as negative regulators of actin polymerization. Neutrophils with pharmacologically inhibited oxidase or isolated from Chronic Granulomatous Disease (CGD) patient and mice displayed enhanced F-actin polymerization, multiple pseudopods formation and impaired chemotaxis. ROS localized to pseudopodia and inhibited actin polymerization by driving actin glutathionylation at the leading edge of migrating cells. Consistent with these in vitro results, adoptively transferred CGD murine neutrophils also showed impaired in vivo recruitment to sites of inflammation. Together, these results present a novel physiological role for ROS in regulation of action polymerization and shed new light on the pathogenesis of CGD.

scholarly journals Generation and Characterization of Telomere Length Maintenance in Tankyrase 2-Deficient Mice

2006 ◽  
Vol 26 (6) ◽  
pp. 2037-2043 ◽  
Author(s):  
Y. Jeffrey Chiang ◽  
My-Linh Nguyen ◽  
Sujatha Gurunathan ◽  
Patrick Kaminker ◽  
Lino Tessarollo ◽  
...  
Keyword(s):  
Telomere Length ◽  
Germ Line ◽  
Mammalian Species ◽  
Polymerase Activity ◽  
Binding Activity ◽  
Telomere Elongation ◽  
Associated Proteins ◽  
And Function ◽  
Tankyrase 1

ABSTRACT Telomere length and function are crucial factors that determine the capacity for cell proliferation and survival, mediate cellular senescence, and play a role in malignant transformation in eukaryotic systems. The telomere length of a specific mammalian species is maintained within a given range by the action of telomerase and telomere-associated proteins. TRF1 is a telomere-associated protein that inhibits telomere elongation by its binding to telomere repeats, preventing access to telomerase. Human TRF1 interacts with tankyrase 1 and tankyrase 2 proteins, two related members of the tankyrase family shown to have poly(ADP-ribose) polymerase activity. Human tankyrase 1 is reported to ADP-ribosylate TRF1 and to down-regulate the telomeric repeat binding activity of TRF1, resulting in telomerase-dependent telomere elongation. Human tankyrase 2 is proposed to have activity similar to that of tankyrase 1, although tankyrase 2 function has been less extensively characterized. In the present study, we have assessed the in vivo function of mouse tankyrase 2 by germ line gene inactivation and show that inactivation of tankyrase 2 does not result in detectable alteration in telomere length when monitored through multiple generations of breeding. This finding suggests that either mouse tankyrases 1 and 2 have redundant functions in telomere length maintenance or that mouse tankyrase 2 differs from human tankyrase 2 in its role in telomere length maintenance. Tankyrase 2 deficiency did result in a significant decrease in body weight sustained through at least the first year of life, most marked in male mice, suggesting that tankyrase 2 functions in potentially telomerase-independent pathways to affect overall development and/or metabolism.

scholarly journals New insights into cadherin function in epidermal sheet formation and maintenance of tissue integrity

2008 ◽  
Vol 105 (40) ◽  
pp. 15405-15410 ◽  
Author(s):  
Christopher L. Tinkle ◽  
H. Amalia Pasolli ◽  
Nicole Stokes ◽  
Elaine Fuchs
Keyword(s):  
Adherens Junction ◽  
Actin Dynamics ◽  
Loss Of Function ◽  
Cortical Actin ◽  
Tissue Integrity ◽  
Gene Ablation ◽  
Rnai Technology ◽  
Physiological Relevance

Co-expression and gene linkage have hampered elucidating the physiological relevance of cadherins in mammalian tissues. Here, we combine conditional gene ablation and transgenic RNA interference to uncover new roles for E- and P-cadherins in epidermal sheet formation in vitro and maintenance of epidermal integrity in vivo. By devising skin-specific RNAi technology, we demonstrate that cadherin inhibition in vivo impairs junction formation and intercellular adhesion and increases apoptosis. These defects compromise epidermal barrier function and tissue integrity. In vitro, with only E-cadherin missing, epidermal sheet formation is delayed, but when both cadherins are suppressed, defects extend to adherens junctions, desmosomes, tight junctions and cortical actin dynamics. Using different rescue strategies, we show that cadherin level rather than subtype is critical. Finally, by comparing conditional loss-of-function studies of epidermal catenins and cadherins, we dissect cadherin-dependent and independent roles of adherens junction components in tissue physiology.

scholarly journals ZapA tetramerization is required for midcell localization and ZapB interaction in Escherichia coli

2019 ◽  
Author(s):  
Nils Y. Meiresonne ◽  
Tanneke den Blaauwen
Keyword(s):  
Cell Division ◽  
Chromosome Segregation ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bacterial Cell Division ◽  
Cellular Processes ◽  
Interaction Partners ◽  
Associated Proteins

AbstractBacterial cell division is guided by FtsZ treadmilling precisely at midcell. FtsZ itself is regulated by FtsZ associated proteins (Zaps) that couple it to different cellular processes. ZapA is known to enhance FtsZ bundling but also forms the synchronizing link with chromosome segregation through ZapB and matS bound MatP. ZapA exists as dimers and tetramers in the cell. Using the ZapAI83E mutant that only forms dimers, this paper investigates the effects of ZapA multimerization state on its interaction partners and cell division. By employing (fluorescence) microscopy and Förster Resonance Energy Transfer in vivo it is shown that; dimeric ZapA is unable to complement a zapA deletion strain and localizes diffusely through the cell but still interacts with FtsZ that is not part of the cell division machinery. Dimeric ZapA is unable to recruit ZapB, which localizes in its presence unipolarly in the cell. Interestingly, the localization profiles of the chromosome and unipolar ZapB anticorrelate. The work presented here confirms previously reported in vitro effects of ZapA multimerization in vivo and further places it in a broader context by revealing the strong implications for ZapB localization and ter linkage.

scholarly journals Effects of mutating α-tubulin lysine 40 on sensory dendrite development

2017 ◽  
Author(s):  
Brian V. Jenkins ◽  
Harriet A. J. Saunders ◽  
Helena L. Record ◽  
Dena M. Johnson-Schlitz ◽  
Jill Wildonger
Keyword(s):  
Targeted Mutagenesis ◽  
Neuronal Structure ◽  
Structural Requirement ◽  
Neuronal Morphogenesis ◽  
Dendrite Development ◽  
Dendrite Branch ◽  
Associated Proteins ◽  
And Function ◽  
Dendrite Morphogenesis

ABSTRACTMicrotubules are essential to neuronal structure and function. Axonal and dendritic microtubules are enriched in post-translational modifications that impact microtubule dynamics, transport, and microtubule-associated proteins. Acetylation of α-tubulin lysine 40 (K40) is a prominent, conserved modification of neuronal microtubules. However, the cellular role of microtubule acetylation remains controversial. To resolve how microtubule acetylation might affect neuronal morphogenesis we mutated endogenous α-tubulin in vivo using a new fly strain that facilitates the rapid knock-in of designer α-tubulin alleles. Leveraging our new strain, we found that microtubule acetylation, as well as polyglutamylation and (de)tyrosination, is not essential for survival. However, we found that dendrite branch refinement in sensory neurons relies on α-tubulin K40. Mutagenesis of K40 reveals moderate yet significant changes in dendritic lysosome transport, microtubule polymerization, and Futsch distribution in dendrites but not axons. Our studies point to an unappreciated role for α-tubulin K40 and acetylation in dendrite morphogenesis. While our results are consistent with the idea that microtubule acetylation patterns microtubule function within neurons, they also suggest there may be a structural requirement for α-tubulin K40.Summary StatementNeurons are enriched in post-translationally modified microtubules. Targeted mutagenesis of endogenous α-tubulin in flies reveals that dendrite branch refinement is altered by acetylation-blocking mutations.

scholarly journals Protein manipulation using single copies of short peptide tags in cultured cells and in Drosophila melanogaster

2020 ◽  
Author(s):  
M. Alessandra Vigano ◽  
Clara-Maria Ell ◽  
Manuela MM Kustermann ◽  
Gustavo Aguilar ◽  
Shinya Matsuda ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Single Copy ◽  
Specific Protein ◽  
Cellular Functions ◽  
Cellular Processes ◽  
Peptide Tags ◽  
Genetic Technologies ◽  
Protein Binders ◽  
And Function

AbstractCellular development and specialized cellular functions are regulated processes which rely on highly dynamic molecular interactions among proteins, distributed in all cell compartments. Analysis of these interactions and their mechanisms of action has been one of the main topics in cellular and developmental research over the last fifty years. Studying and understanding the functions of proteins of interest (POIs) has been mostly achieved by their alteration at the genetic level and the analysis of the phenotypic changes generated by these alterations. Although genetic and reverse genetic technologies contributed to the vast majority of information and knowledge we have gathered so far, targeting specific interactions of POIs in a time- and space-controlled manner or analyzing the role of POIs in dynamic cellular processes such as cell migration or cell division would require more direct approaches. The recent development of specific protein binders, which can be expressed and function intracellularly, together with several improvements in synthetic biology techniques, have contributed to the creation of a new toolbox for direct protein manipulations. We selected a number of short tag epitopes for which protein binders from different scaffolds have been developed and tested whether these tags can be bound by the corresponding protein binders in living cells when they are inserted in a single copy in a POI. We indeed find that in all cases, a single copy of a short tag allows protein binding and manipulation. Using Drosophila, we also find that single short tags can be recognized and allow degradation and relocalization of POIs in vivo.

scholarly journals Protein manipulation using single copies of short peptide tags in cultured cells and in Drosophila melanogaster

Development ◽  
2021 ◽  
Vol 148 (6) ◽  
Author(s):  
M. Alessandra Vigano ◽  
Clara-Maria Ell ◽  
Manuela M. M. Kustermann ◽  
Gustavo Aguilar ◽  
Shinya Matsuda ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Specific Protein ◽  
Cellular Processes ◽  
Cell Compartments ◽  
Peptide Tags ◽  
Genetic Level ◽  
Protein Binders ◽  
And Function

ABSTRACT Cellular development and function rely on highly dynamic molecular interactions among proteins distributed in all cell compartments. Analysis of these interactions has been one of the main topics in cellular and developmental research, and has been mostly achieved by the manipulation of proteins of interest (POIs) at the genetic level. Although genetic strategies have significantly contributed to our current understanding, targeting specific interactions of POIs in a time- and space-controlled manner or analysing the role of POIs in dynamic cellular processes, such as cell migration or cell division, would benefit from more-direct approaches. The recent development of specific protein binders, which can be expressed and function intracellularly, along with advancement in synthetic biology, have contributed to the creation of a new toolbox for direct protein manipulations. Here, we have selected a number of short-tag epitopes for which protein binders from different scaffolds have been generated and showed that single copies of these tags allowed efficient POI binding and manipulation in living cells. Using Drosophila, we also find that single short tags can be used for POI manipulation in vivo.

scholarly journals Structure of the actin-depolymerizing factor homology domain in complex with actin

2008 ◽  
Vol 182 (1) ◽  
pp. 51-59 ◽  
Author(s):  
Ville O. Paavilainen ◽  
Esko Oksanen ◽  
Adrian Goldman ◽  
Pekka Lappalainen
Keyword(s):  
Crystal Structure ◽  
Atomic Structure ◽  
Actin Filaments ◽  
Driving Force ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Actin Monomer ◽  
Nucleotide Exchange ◽  
Actin Depolymerizing Factor ◽  
Cellular Processes

Actin dynamics provide the driving force for many cellular processes including motility and endocytosis. Among the central cytoskeletal regulators are actin-depolymerizing factor (ADF)/cofilin, which depolymerizes actin filaments, and twinfilin, which sequesters actin monomers and caps filament barbed ends. Both interact with actin through an ADF homology (ADF-H) domain, which is also found in several other actin-binding proteins. However, in the absence of an atomic structure for the ADF-H domain in complex with actin, the mechanism by which these proteins interact with actin has remained unknown. Here, we present the crystal structure of twinfilin's C-terminal ADF-H domain in complex with an actin monomer. This domain binds between actin subdomains 1 and 3 through an interface that is conserved among ADF-H domain proteins. Based on this structure, we suggest a mechanism by which ADF/cofilin and twinfilin inhibit nucleotide exchange of actin monomers and present a model for how ADF/cofilin induces filament depolymerization by weakening intrafilament interactions.

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