scholarly journals The WASP Homologue Las17 Activates the Novel Actin-regulatory Activity of Ysc84 to Promote Endocytosis in Yeast

2009 ◽  
Vol 20 (6) ◽  
pp. 1618-1628 ◽  
Author(s):  
Alastair S. Robertson ◽  
Ellen G. Allwood ◽  
Adam P.C. Smith ◽  
Fiona C. Gardiner ◽  
Rosaria Costa ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Actin Polymerization ◽  
Actin Binding ◽  
The Novel ◽  
Cellular Processes ◽  
New Class ◽  
Actin Binding Domain ◽  
Kinetics Of ◽  
Actin Binding Site

Actin plays an essential role in many eukaryotic cellular processes, including motility, generation of polarity, and membrane trafficking. Actin function in these roles is regulated by association with proteins that affect its polymerization state, dynamics, and organization. Numerous proteins have been shown to localize with cortical patches of yeast actin during endocytosis, but the role of many of these proteins remains poorly understood. Here, we reveal that the yeast protein Ysc84 represents a new class of actin-binding proteins, conserved from yeast to humans. It contains a novel N-terminal actin-binding domain termed Ysc84 actin binding (YAB), which can bind and bundle actin filaments. Intriguingly, full-length Ysc84 alone does not bind to actin, but binding can be activated by a specific motif within the polyproline region of the yeast WASP homologue Las17. We also identify a new monomeric actin-binding site on Las17. Together, the polyproline region of Las17 and Ysc84 can promote actin polymerization. Using live cell imaging, kinetics of assembly and disassembly of proteins at the endocytic site were analyzed and reveal that loss of Ysc84 and its homologue Lsb3 decrease inward movement of vesicles consistent with a role in actin polymerization during endocytosis.

The Actin Regulator Coronin-1A Modulates Platelet Shape Change and Consolidates Arterial Thrombosis

2018 ◽  
Vol 118 (12) ◽  
pp. 2098-2111 ◽  
Author(s):  
Thomas Stocker ◽  
Joachim Pircher ◽  
Artid Skenderi ◽  
Andreas Ehrlich ◽  
Clemens Eberle ◽  
...  
Keyword(s):  
Arterial Thrombosis ◽  
Actin Polymerization ◽  
Thrombus Formation ◽  
Shape Change ◽  
Actin Binding ◽  
Cellular Processes ◽  
Reduced Density ◽  
Induced Aggregation ◽  
Platelet Shape

AbstractCoronin-1A (Coro1A) belongs to a family of highly conserved actin-binding proteins that regulate cytoskeletal re-arrangement. In mammalians, Coro1A expression is most abundant in the haematopoietic lineage, where it regulates various cellular processes. The role of Coro1A in platelets has been previously unknown. Here, we identified Coro1A in human and mouse platelets. Genetic absence of Coro1A in mouse platelets inhibited agonist-induced actin polymerization and altered cofilin phosphoregulation, leading to a reduction in spreading and low-dose collagen induced aggregation. Furthermore, Coro1A-deficient mice displayed a defect in ferric chloride-induced arterial thrombosis with prolonged thrombus formation and reduced thrombus size. Immunofluorescence analysis revealed a less compact thrombus structure with reduced density of platelets and fibrinogen. In summary, Coro1A has a role in platelet biology with impact on spreading, aggregation and thrombosis.

scholarly journals The Central Role of the F-Actin Surface in Myosin Force Generation

Biology ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 1221
Author(s):  
Matthew H. Doran ◽  
William Lehman
Keyword(s):  
Critical Role ◽  
Structural Features ◽  
Force Generation ◽  
Actin Binding ◽  
Myosin Isoforms ◽  
Thin Filaments ◽  
Cellular Processes ◽  
Muscle Myosin ◽  
Kinetics Of

Actin is one of the most abundant and versatile proteins in eukaryotic cells. As discussed in many contributions to this Special Issue, its transition from a monomeric G-actin to a filamentous F-actin form plays a critical role in a variety of cellular processes, including control of cell shape and cell motility. Once polymerized from G-actin, F-actin forms the central core of muscle-thin filaments and acts as molecular tracks for myosin-based motor activity. The ATP-dependent cross-bridge cycle of myosin attachment and detachment drives the sliding of myosin thick filaments past thin filaments in muscle and the translocation of cargo in somatic cells. The variation in actin function is dependent on the variation in muscle and non-muscle myosin isoform behavior as well as interactions with a plethora of additional actin-binding proteins. Extensive work has been devoted to defining the kinetics of actin-based force generation powered by the ATPase activity of myosin. In addition, over the past decade, cryo-electron microscopy has revealed the atomic-evel details of the binding of myosin isoforms on the F-actin surface. Most accounts of the structural interactions between myosin and actin are described from the perspective of the myosin molecule. Here, we discuss myosin-binding to actin as viewed from the actin surface. We then describe conserved structural features of actin required for the binding of all or most myosin isoforms while also noting specific interactions unique to myosin isoforms.

scholarly journals Inhibition of actin polymerization by a synthetic dodecapeptide patterned on the sequence around the actin-binding site of cofilin

1991 ◽  
Vol 266 (16) ◽  
pp. 10485-10489
Author(s):  
N. Yonezawa ◽  
E. Nishida ◽  
K. Iida ◽  
H. Kumagai ◽  
I. Yahara ◽  
...  
Keyword(s):  
Binding Site ◽  
Actin Polymerization ◽  
Actin Binding ◽  
Actin Binding Site

scholarly journals Dynamics of Tpm1.8 domains on actin filaments with single molecule resolution

2020 ◽  
Author(s):  
Ilina Bareja ◽  
Hugo Wioland ◽  
Miro Janco ◽  
Philip R. Nicovich ◽  
Antoine Jégou ◽  
...  
Keyword(s):  
Actin Filament ◽  
Single Molecule ◽  
Actin Filaments ◽  
High Probability ◽  
Actin Binding ◽  
Single Molecule Level ◽  
Filament Dynamics ◽  
Kinetics Of ◽  
Insight Into

ABSTRACTTropomyosins regulate dynamics and functions of the actin cytoskeleton by forming long chains along the two strands of actin filaments that act as gatekeepers for the binding of other actin-binding proteins. The fundamental molecular interactions underlying the binding of tropomyosin to actin are still poorly understood. Using microfluidics and fluorescence microscopy, we observed the binding of fluorescently labelled tropomyosin isoform Tpm1.8 to unlabelled actin filaments in real time. This approach in conjunction with mathematical modeling enabled us to quantify the nucleation, assembly and disassembly kinetics of Tpm1.8 on single filaments and at the single molecule level. Our analysis suggests that Tpm1.8 decorates the two strands of the actin filament independently. Nucleation of a growing tropomyosin domain proceeds with high probability as soon as the first Tpm1.8 molecule is stabilised by the addition of a second molecule, ultimately leading to full decoration of the actin filament. In addition, Tpm1.8 domains are asymmetrical, with enhanced dynamics at the edge oriented towards the barbed end of the actin filament. The complete description of Tpm1.8 kinetics on actin filaments presented here provides molecular insight into actin-tropomyosin filament formation and the role of tropomyosins in regulating actin filament dynamics.

scholarly journals Coupling of the nucleus and cytoplasm: Role of the LINC complex

2005 ◽  
Vol 172 (1) ◽  
pp. 41-53 ◽  
Author(s):  
Melissa Crisp ◽  
Qian Liu ◽  
Kyle Roux ◽  
J.B. Rattner ◽  
Catherine Shanahan ◽  
...  
Keyword(s):  
Nuclear Membrane ◽  
Integral Membrane Proteins ◽  
Actin Binding ◽  
Linc Complex ◽  
Outer Membranes ◽  
Perinuclear Space ◽  
Actin Binding Domain ◽  
Lumenal Domain ◽  
Integral Proteins

The nuclear envelope defines the barrier between the nucleus and cytoplasm and features inner and outer membranes separated by a perinuclear space (PNS). The inner nuclear membrane contains specific integral proteins that include Sun1 and Sun2. Although the outer nuclear membrane (ONM) is continuous with the endoplasmic reticulum, it is nevertheless enriched in several integral membrane proteins, including nesprin 2 Giant (nesp2G), an 800-kD protein featuring an NH2-terminal actin-binding domain. A recent study (Padmakumar, V.C., T. Libotte, W. Lu, H. Zaim, S. Abraham, A.A. Noegel, J. Gotzmann, R. Foisner, and I. Karakesisoglou. 2005. J. Cell Sci. 118:3419–3430) has shown that localization of nesp2G to the ONM is dependent upon an interaction with Sun1. In this study, we confirm and extend these results by demonstrating that both Sun1 and Sun2 contribute to nesp2G localization. Codepletion of both of these proteins in HeLa cells leads to the loss of ONM-associated nesp2G, as does overexpression of the Sun1 lumenal domain. Both treatments result in the expansion of the PNS. These data, together with those of Padmakumar et al. (2005), support a model in which Sun proteins tether nesprins in the ONM via interactions spanning the PNS. In this way, Sun proteins and nesprins form a complex that links the nucleoskeleton and cytoskeleton (the LINC complex).

scholarly journals Mammalian inositol polyphosphate 5-phosphatase II can compensate for the absence of all three yeast Sac1-like-domain-containing 5-phosphatases

2001 ◽  
Vol 355 (3) ◽  
pp. 805-817 ◽  
Author(s):  
Cindy J. O'MALLEY ◽  
Brad K. McCOLL ◽  
Anne M. KONG ◽  
Sarah L. ELLIS ◽  
A. Primrose W. WIJAYARATNAM ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Inducible Promoter ◽  
Actin Binding ◽  
Inositol Polyphosphate ◽  
Null Mutant ◽  
Phosphatase Domain ◽  
Signalling Molecules ◽  
Yeast Strains ◽  
Bud Site

Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] plays a complex role in generating intracellular signalling molecules, and also in regulating actin-binding proteins, vesicular trafficking and vacuolar fusion. Four inositol polyphosphate 5-phosphatases (hereafter called 5-phosphatases) have been identified in Saccharomyces cerevisiae: Inp51p, Inp52p, Inp53p and Inp54p. Each enzyme contains a 5-phosphatase domain which hydrolyses PtdIns(4,5)P2, forming PtdIns4P, while Inp52p and Inp53p also express a polyphosphoinositide phosphatase domain within the Sac1-like domain. Disruption of any two yeast 5-phosphatases containing a Sac1-like domain results in abnormalities in actin polymerization, plasma membrane, vacuolar morphology and bud-site selection. Triple null mutant 5-phosphatase strains are non-viable. To investigate the role of PtdIns(4,5)P2 in mediating the phenotype of double and triple 5-phosphatase null mutant yeast, we determined whether a mammalian PtdIns(4,5)P2 5-phosphatase, 5-phosphatase II, which lacks polyphosphoinositide phosphatase activity, could correct the phenotype of triple 5-phosphatase null mutant yeast and restore cellular PtdIns(4,5)P2 levels to near basal values. Mammalian 5-phosphatase II expressed under an inducible promoter corrected the growth, cell wall, vacuolar and actin polymerization defects of the triple 5-phosphatase null mutant yeast strains. Cellular PtdIns(4,5)P2 levels in various 5-phosphatase double null mutant strains demonstrated significant accumulation (4.5-, 3- and 2-fold for ∆inp51∆inp53, ∆inp51∆inp52 and ∆inp52∆inp53 double null mutants respectively), which was corrected significantly following 5-phosphatase II expression. Collectively, these studies demonstrate the functional and cellular consequences of PtdIns(4,5)P2 accumulation and the evolutionary conservation of function between mammalian and yeast PtdIns(4,5)P2 5-phosphatases.

scholarly journals A New Class of Repression Modules Is Critical for Heme Regulation of the Yeast Transcriptional Activator Hap1

1999 ◽  
Vol 19 (6) ◽  
pp. 4324-4333 ◽  
Author(s):  
Angela Hach ◽  
Thomas Hon ◽  
Li Zhang
Keyword(s):  
Biochemical Analysis ◽  
Activator Protein 1 ◽  
Heme Binding ◽  
Systematic Analysis ◽  
High Molecular Weight Complex ◽  
Cellular Processes ◽  
New Class ◽  
Heme Domain ◽  
Heme Regulation

ABSTRACTHeme plays key regulatory roles in numerous molecular and cellular processes for systems that sense or use oxygen. In the yeastSaccharomyces cerevisiae, oxygen sensing and heme signaling are mediated by heme activator protein 1 (Hap1). Hap1 contains seven heme-responsive motifs (HRMs): six are clustered in the heme domain, and a seventh is near the activation domain. To determine the functional role of HRMs and to define which parts of Hap1 mediate heme regulation, we carried out a systematic analysis of Hap1 mutants with various regions deleted or mutated. Strikingly, the data show that HRM1 to -6, located in the previously designated Hap1 heme domain, have little impact on heme regulation. All seven HRMs are dispensable for Hap1 repression in the absence of heme, but HRM7 is required for Hap1 activation by heme. More importantly, we show that a novel class of repression modules—RPM1, encompassing residues 245 to 278; RPM2, encompassing residues 1061 to 1185; and RPM3, encompassing residues 203 to 244—is critical for Hap1 repression in the absence of heme. Biochemical analysis indicates that RPMs mediate Hap1 repression, at least partly, by the formation of a previously identified higher-order complex termed the high-molecular-weight complex (HMC), while HRMs mediate heme activation by permitting heme binding and the disassembly of the HMC. These findings provide significant new insights into the molecular interactions critical for Hap1 repression in the absence of heme and Hap1 activation by heme.

scholarly journals EPLIN regulates actin dynamics by cross-linking and stabilizing filaments

2003 ◽  
Vol 160 (3) ◽  
pp. 399-407 ◽  
Author(s):  
Raymond S. Maul ◽  
Yuhong Song ◽  
Kurt J. Amann ◽  
Sachi C. Gerbin ◽  
Thomas D. Pollard ◽  
...  
Keyword(s):  
Actin Filament ◽  
Actin Polymerization ◽  
Binding Activity ◽  
Stress Fibers ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Membrane Ruffling ◽  
Cross Links ◽  
As Stress ◽  
Kinetics Of

Epithelial protein lost in neoplasm (EPLIN) is a cytoskeleton-associated protein encoded by a gene that is down-regulated in transformed cells. EPLIN increases the number and size of actin stress fibers and inhibits membrane ruffling induced by Rac. EPLIN has at least two actin binding sites. Purified recombinant EPLIN inhibits actin filament depolymerization and cross-links filaments in bundles. EPLIN does not affect the kinetics of spontaneous actin polymerization or elongation at the barbed end, but inhibits branching nucleation of actin filaments by Arp2/3 complex. Side binding activity may stabilize filaments and account for the inhibition of nucleation mediated by Arp2/3 complex. We propose that EPLIN promotes the formation of stable actin filament structures such as stress fibers at the expense of more dynamic actin filament structures such as membrane ruffles. Reduced expression of EPLIN may contribute to the motility of invasive tumor cells.

scholarly journals Disruption of the utrophin–actin interaction by monoclonal antibodies and prediction of an actin-binding surface of utrophin

1998 ◽  
Vol 337 (1) ◽  
pp. 119-123 ◽  
Author(s):  
Glenn E. MORRIS ◽  
Nguyen thi MAN ◽  
Nguyen thi Ngoc HUYEN ◽  
Alexander PEREBOEV ◽  
John KENDRICK-JONES ◽  
...  
Keyword(s):  
Binding Sites ◽  
Three Dimensional ◽  
Binding Domain ◽  
Actin Binding ◽  
Dimensional Structure ◽  
Functional Regions ◽  
Binding Surface ◽  
Actin Binding Domain ◽  
Actin Binding Site ◽  
Helical Region

Monoclonal antibody (mAb) binding sites in the N-terminal actin-binding domain of utrophin have been identified using phage-displayed peptide libraries, and the mAbs have been used to probe functional regions of utrophin involved in actin binding. mAbs were characterized for their ability to interact with the utrophin actin-binding domain and to affect actin binding to utrophin in sedimentation assays. One of these antibodies was able to inhibit utrophin–F-actin binding and was shown to recognize a predicted helical region at residues 13–22 of utrophin, close to a previously predicted actin-binding site. Two other mAbs which did not affect actin binding recognized predicted loops in the second calponin homology domain of the utrophin actin-binding domain. Using the known three-dimensional structure of the homologous actin-binding domain of fimbrin, these results have enabled us to determine the likely orientation of the utrophin actin-binding domain with respect to the actin filament.

Aldolase-localization in cultured cells: Cell-type and substrate-specific regulation of cytoskeletal associations

2001 ◽  
Vol 79 (6) ◽  
pp. 719-728 ◽  
Author(s):  
Ralf Schindler ◽  
Elke Weichselsdorfer ◽  
Oliver Wagner ◽  
Jürgen Bereiter-Hahn
Keyword(s):  
Electron Microscopy ◽  
Intermediate Filaments ◽  
Actin Polymerization ◽  
Cultured Cells ◽  
Actin Binding ◽  
Permeabilized Cells ◽  
Specific Regulation ◽  
Actin Fibrils ◽  
Fructose 1,6 Bisphosphate

The role of aldolase as a true F- and G-actin binding protein, including modulating actin polymerization, initiating bundling, and giving rise to supramolecular structures that emanate from actin fibrils, has been established using indirect immunofluorescence, permeabilization of XTH-2 cells and keratocytes, and microinjection of fluorescence-labeled aldolase. In addition, binding to intermediate filaments, vimentin, and cytokeratins has been demonstrated. In permeabilized cells in the presence of fructose-1,6-bisphosphate (20–2000 µM) aldolase shifts from association with actin fibres to intermediate filaments. Plenty of free binding sites on microtubules have been revealed by addition of fluorochromed aldolase derived from rabbit skeletal muscle. However, endogenous aldolase was never found associated with microtubules. Differences in actin polymerization in the presence of aldolase as revealed by pyrene-labeled actin fluorimetry and viscosimetry were explained by electron microscopy showing the formation of rod-like structures (10 nm wide, 20–60 nm in length) by association of aldolase with G-actin, which prevents further polymerization. Upon the addition of fructose-1,6-bisphosphate, G-actin–aldolase mixture polymerizes to a higher viscosity and forms stiffer filaments than pure actin of the same concentration.Key words: aldolase, cytoskeleton, electron microscopy, viscosimetry.

Sign in / Sign up

Export Citation Format

Share Document