scholarly journals Interaction of Sla2p's ANTH Domain with PtdIns(4,5)P2 Is Important for Actin-dependent Endocytic Internalization

2005 ◽  
Vol 16 (2) ◽  
pp. 717-730 ◽  
Author(s):  
Yidi Sun ◽  
Marko Kaksonen ◽  
David T. Madden ◽  
Randy Schekman ◽  
David G. Drubin
Keyword(s):  
Cell Growth ◽  
Fluorescence Microscopy ◽  
Particle Tracking ◽  
Binding Assay ◽  
Actin Binding ◽  
Cell Cortex ◽  
Actin Organization ◽  
Lysine Residues ◽  
Endocytic Machinery

A variety of studies have implicated the lipid PtdIns(4,5)P2 in endocytic internalization, but how this lipid mediates its effects is not known. The AP180 N-terminal homology (ANTH) domain is a PtdIns(4,5)P2-binding module found in several proteins that participate in receptor-mediated endocytosis. One such protein is yeast Sla2p, a highly conserved actin-binding protein essential for actin organization and endocytic internalization. To better understand how PtdIns(4,5)P2 binding regulates actin-dependent endocytosis, we investigated the functions of Sla2p's ANTH domain. A liposome-binding assay revealed that Sla2p binds to PtdIns(4,5)P2 specifically through its ANTH domain and identified specific lysine residues required for this interaction. Mutants of Sla2p deficient in PtdIns(4,5)P2 binding showed significant defects in cell growth, actin organization, and endocytic internalization. These defects could be rescued by increasing PtdIns(4,5)P2 levels in vivo. Strikingly, mutant Sla2p defective in PtdIns(4,5)P2 binding localized with the endocytic machinery at the cell cortex, establishing that the ANTH-PtdIns(4,5)P2 interaction is not necessary for this association. In contrast, multicolor real-time fluorescence microscopy and particle-tracking analysis demonstrated that PtdIns(4,5)P2 binding is required during endocytic internalization. These results demonstrate that the interaction of Sla2p's ANTH domain with PtdIns(4,5)P2 plays a key role in regulation of the dynamics of actin-dependent endocytic internalization.

scholarly journals A High-affinity Interaction with ADP-Actin Monomers Underlies the Mechanism and In Vivo Function of Srv2/cyclase-associated Protein

2004 ◽  
Vol 15 (11) ◽  
pp. 5158-5171 ◽  
Author(s):  
Pieta K. Mattila ◽  
Omar Quintero-Monzon ◽  
Jamie Kugler ◽  
James B. Moseley ◽  
Steven C. Almo ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Actin Binding ◽  
Actin Monomer ◽  
Nucleotide Exchange ◽  
Actin Organization ◽  
C Terminus ◽  
Actin Binding Domain ◽  
Affinity Interaction

Cyclase-associated protein (CAP), also called Srv2 in Saccharomyces cerevisiae, is a conserved actin monomer-binding protein that promotes cofilin-dependent actin turnover in vitro and in vivo. However, little is known about the mechanism underlying this function. Here, we show that S. cerevisiae CAP binds with strong preference to ADP-G-actin (Kd 0.02 μM) compared with ATP-G-actin (Kd 1.9 μM) and competes directly with cofilin for binding ADP-G-actin. Further, CAP blocks actin monomer addition specifically to barbed ends of filaments, in contrast to profilin, which blocks monomer addition to pointed ends of filaments. The actin-binding domain of CAP is more extensive than previously suggested and includes a recently solved β-sheet structure in the C-terminus of CAP and adjacent sequences. Using site-directed mutagenesis, we define evolutionarily conserved residues that mediate binding to ADP-G-actin and demonstrate that these activities are required for CAP function in vivo in directing actin organization and polarized cell growth. Together, our data suggest that in vivo CAP competes with cofilin for binding ADP-actin monomers, allows rapid nucleotide exchange to occur on actin, and then because of its 100-fold weaker binding affinity for ATP-actin compared with ADP-actin, allows other cellular factors such as profilin to take the handoff of ATP-actin and facilitate barbed end assembly.

scholarly journals In Vivo Importance of Actin Nucleotide Exchange Catalyzed by Profilin

2000 ◽  
Vol 150 (4) ◽  
pp. 895-904 ◽  
Author(s):  
Amy K. Wolven ◽  
Lisa D. Belmont ◽  
Nicole M. Mahoney ◽  
Steven C. Almo ◽  
David G. Drubin
Keyword(s):  
Point Mutations ◽  
Fluid Phase ◽  
Intrinsic Rate ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Nucleotide Exchange ◽  
Actin Organization ◽  
Cytoskeleton Organization

The actin monomer-binding protein, profilin, influences the dynamics of actin filaments in vitro by suppressing nucleation, enhancing nucleotide exchange on actin, and promoting barbed-end assembly. Profilin may also link signaling pathways to actin cytoskeleton organization by binding to the phosphoinositide PIP2 and to polyproline stretches on several proteins. Although activities of profilin have been studied extensively in vitro, the significance of each of these activities in vivo needs to be tested. To study profilin function, we extensively mutagenized the Saccharomyces cerevisiae profilin gene (PFY1) and examined the consequences of specific point mutations on growth and actin organization. The actin-binding region of profilin was shown to be critical in vivo. act1-157, an actin mutant with an increased intrinsic rate of nucleotide exchange, suppressed defects in actin organization, cell growth, and fluid-phase endocytosis of pfy1-4, a profilin mutant defective in actin binding. In reactions containing actin, profilin, and cofilin, profilin was required for fast rates of actin filament turnover. However, Act1-157p circumvented the requirement for profilin. Based on the results of these studies, we conclude that in living cells profilin promotes rapid actin dynamics by regenerating ATP actin from ADP actin–cofilin generated during filament disassembly.

scholarly journals The actin-binding protein Hip1R associates with clathrin during early stages of endocytosis and promotes clathrin assembly in vitro

2001 ◽  
Vol 154 (6) ◽  
pp. 1209-1224 ◽  
Author(s):  
Åsa E.Y. Engqvist-Goldstein ◽  
Robin A. Warren ◽  
Michael M. Kessels ◽  
James H. Keen ◽  
John Heuser ◽  
...  
Keyword(s):  
Binding Protein ◽  
Coiled Coil ◽  
Actin Binding ◽  
Live Cells ◽  
Cell Cortex ◽  
Coated Pits ◽  
Actin Binding Protein ◽  
Real Time Analysis

Huntingtin-interacting protein 1 related (Hip1R) is a novel component of clathrin-coated pits and vesicles and is a mammalian homologue of Sla2p, an actin-binding protein important for both actin organization and endocytosis in yeast. Here, we demonstrate that Hip1R binds via its putative central coiled-coil domain to clathrin, and provide evidence that Hip1R and clathrin are associated in vivo at sites of endocytosis. First, real-time analysis of Hip1R–YFP and DsRed–clathrin light chain (LC) in live cells revealed that these proteins show almost identical temporal and spatial regulation at the cell cortex. Second, at the ultrastructure level, immunogold labeling of ‘unroofed’ cells showed that Hip1R localizes to clathrin-coated pits. Third, overexpression of Hip1R affected the subcellular distribution of clathrin LC. Consistent with a functional role for Hip1R in endocytosis, we also demonstrated that it promotes clathrin cage assembly in vitro. Finally, we showed that Hip1R is a rod-shaped apparent dimer with globular heads at either end, and that it can assemble clathrin-coated vesicles and F-actin into higher order structures. In total, Hip1R's properties suggest an early endocytic function at the interface between clathrin, F-actin, and lipids.

scholarly journals The actin-binding ERM protein Moesin binds to and stabilizes microtubules at the cell cortex

2013 ◽  
Vol 202 (2) ◽  
pp. 251-260 ◽  
Author(s):  
Sara Solinet ◽  
Kazi Mahmud ◽  
Shannon F. Stewman ◽  
Khaled Ben El Kadhi ◽  
Barbara Decelle ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Metastatic Potential ◽  
Actin Binding ◽  
Cell Cortex ◽  
Shape Transformation ◽  
Erm Proteins ◽  
Cellular Processes ◽  
Anaphase Onset

Ezrin, Radixin, and Moesin (ERM) proteins play important roles in many cellular processes including cell division. Recent studies have highlighted the implications of their metastatic potential in cancers. ERM’s role in these processes is largely attributed to their ability to link actin filaments to the plasma membrane. In this paper, we show that the ERM protein Moesin directly binds to microtubules in vitro and stabilizes microtubules at the cell cortex in vivo. We identified two evolutionarily conserved residues in the FERM (4.1 protein and ERM) domains of ERMs that mediated the association with microtubules. This ERM–microtubule interaction was required for regulating spindle organization in metaphase and cell shape transformation after anaphase onset but was dispensable for bridging actin filaments to the metaphase cortex. These findings provide a molecular framework for understanding the complex functional interplay between the microtubule and actin cytoskeletons mediated by ERM proteins in mitosis and have broad implications in both physiological and pathological processes that require ERMs.

scholarly journals Class II formin targeting to the cell cortex by binding PI(3,5)P2 is essential for polarized growth

2012 ◽  
Vol 198 (2) ◽  
pp. 235-250 ◽  
Author(s):  
Peter A.C. van Gisbergen ◽  
Ming Li ◽  
Shu-Zon Wu ◽  
Magdalena Bezanilla
Keyword(s):  
Cell Growth ◽  
Actin Filaments ◽  
De Novo ◽  
Class Ii ◽  
Lipid Binding ◽  
Cell Cortex ◽  
Polarized Growth ◽  
Cortical Actin ◽  
Cortical Localization

Class II formins are key regulators of actin and are essential for polarized plant cell growth. Here, we show that the class II formin N-terminal phosphatase and tensin (PTEN) domain binds phosphoinositide-3,5-bisphosphate (PI(3,5)P2). Replacing the PTEN domain with polypeptides of known lipid-binding specificity, we show that PI(3,5)P2 binding was required for formin-mediated polarized growth. Via PTEN, formin also localized to the cell apex, phragmoplast, and to the cell cortex as dynamic cortical spots. We show that the cortical localization driven by binding to PI(3,5)P2 was required for function. Silencing the kinases that produce PI(3,5)P2 reduced cortical targeting of formin and inhibited polarized growth. We show a subset of cortical formin spots moved in actin-dependent linear trajectories. We observed that the linearly moving subpopulation of cortical formin generated new actin filaments de novo and along preexisting filaments, providing evidence for formin-mediated actin bundling in vivo. Taken together, our data directly link PI(3,5)P2 to generation and remodeling of the cortical actin array.

scholarly journals Exo-endocytic trafficking and the septin-based diffusion barrier are required for the maintenance of Cdc42p polarization during budding yeast asymmetric growth

2011 ◽  
Vol 22 (5) ◽  
pp. 624-633 ◽  
Author(s):  
Kelly Orlando ◽  
Xiaoli Sun ◽  
Jian Zhang ◽  
Tu Lu ◽  
Lauren Yokomizo ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Growth ◽  
Membrane Trafficking ◽  
Daughter Cell ◽  
Lateral Diffusion ◽  
Cell Cortex ◽  
Actin Organization ◽  
Cell Plasma ◽  
Biochemical Fractionation ◽  
Asymmetric Growth

Cdc42p plays a central role in asymmetric cell growth in yeast by controlling actin organization and vesicular trafficking. However, how Cdc42p is maintained specifically at the daughter cell plasma membrane during asymmetric cell growth is unclear. We have analyzed Cdc42p localization in yeast mutants defective in various stages of membrane trafficking by fluorescence microscopy and biochemical fractionation. We found that two separate exocytic pathways mediate Cdc42p delivery to the daughter cell. Defects in one of these pathways result in Cdc42p being rerouted through the other. In particular, the pathway involving trafficking through endosomes may couple Cdc42p endocytosis from, and subsequent redelivery to, the plasma membrane to maintain Cdc42p polarization at the daughter cell. Although the endo-exocytotic coupling is necessary for Cdc42p polarization, it is not sufficient to prevent the lateral diffusion of Cdc42p along the cell cortex. A barrier function conferred by septins is required to counteract the dispersal of Cdc42p and maintain its localization in the daughter cell but has no effect on the initial polarization of Cdc42p at the presumptive budding site before symmetry breaking. Collectively, membrane trafficking and septins function synergistically to maintain the dynamic polarization of Cdc42p during asymmetric growth in yeast.

A Clinically Relevant Zebrafish in Vivo Model of Human Multiple Myeloma (MM) to Study Disease Biology and Preclinical Therapeutic Efficacy

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4078-4078
Author(s):  
Weihong Zhang ◽  
Jianhong Lin ◽  
Dongdong Ma ◽  
Ariel H Kwart ◽  
Aravind Ramachandran ◽  
...  
Keyword(s):  
Cell Growth ◽  
Fluorescence Microscopy ◽  
Drug Treatment ◽  
Cell Survival ◽  
Cell Lines ◽  
Novel Agents ◽  
Post Injection ◽  
Zebrafish Model ◽  
Number Of Cells

Abstract Abstract 4078 Although a number of murine models of human MM have been developed and the SCID-hu murine model allows for growth of primary MM cells, these models have significant limitations including need for insertion of human bones, generation of limited number of mice with human MM Growth and over 2 months lag period, highlighting the need for a model that allows growth of human MM cells rapidly and in larger number of animals. We here describe a zebrafish model which can sustain human patient MM cell growth and be able to test efficacy of therapeutic agents. In this model we have utilized Casper fish, which are transparent and hence ideal for in vivo observation of tumor, Moreover, their 48 hours post fertilization (hpf) stage embryos are immune deficient allowing growth of xenogeneic human MM cells. We have tested growth of both MM cell lines as well as primary patient cells. About 50–200 cells from MM cell lines or CD138+ plasma cells from MM patient bone marrow samples labeled by CM-Dil were injected into the abdominal perivitelline space of 48 hpf stage Casper fish larvae and observed tumor progression, including tumor size and cell invasion by fluorescence microscopy at 24, 48 and 72 hrs post injection. MM cells were confirmed by immunohistochemistry. We observe over 80% larvae demonstrating MM cell survival for up to 9 days. As we require very small number of cells we were able to inject and observe growth of primary cells from all the patients producing over 50 larvae, We next treated the MM cells in vivo by adding first common anti-MM agents into fish water through 24 hrs to 72 hrs post injection (hpi) and observed change in tumor cell survival by fluorescence microscopy again at 24, 48 and 72 hours post treatment (hpt). We have here evaluated response of MM cell lines and MM primary patient cells to single or combination drug treatment, and defined response. We have observed response of the traditional and novel agents as predicted. For example, we have confirmed the response of dexamethasone to MM1.S and MM1.R cell lines which are sensitive or resistant to dexemathasone in this model. We observed that 100 nM dexemathasone treatment led to observation of MM cell survival, and found response ratio of MM1.S and MM1.R xenograft tumors to were 37.5% vs 87.5% fish at 24 hpt and 26.7% vs 87.5% at 48 hpt respectively. We have confirmed efficacy of bortezomib as well as lenalidomide in this model using MM cell lines as well as observed efficacy against primary MM cells. Importantly, we have been able to confirm similar drug resistance profile as patients; for example cells from a patient with bortezomib resistance survived bortezomib treatment in this model. Any combination treatment produced higher response than single drug treatment indicating that MM xenograft zebrafish model can predict drug response accurately. The zebrafish larvaes tolerated all the treatment at the dose utilized well. In conclusion, we have characterized a highly-reproducible zebrafish model of human myeloma that supports primary myeloma cell growth using very small number of cells and can allow generation of large number of fish with evalution of drug activity in a very short time of 3–5 days. This model provides a powerful tool to evaluate preclinical efficacy of novel agents and combinations and may provide opportunity to evaluate personalized therapy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Mammalian Abp1, a Signal-Responsive F-Actin–Binding Protein, Links the Actin Cytoskeleton to Endocytosis via the Gtpase Dynamin

2001 ◽  
Vol 153 (2) ◽  
pp. 351-366 ◽  
Author(s):  
Michael M. Kessels ◽  
Åsa E.Y. Engqvist-Goldstein ◽  
David G. Drubin ◽  
Britta Qualmann
Keyword(s):  
Actin Cytoskeleton ◽  
Binding Protein ◽  
Sh3 Domain ◽  
Actin Binding ◽  
Actin Binding Protein ◽  
Domain Interactions ◽  
Endocytic Machinery ◽  
Transferrin Uptake

The actin cytoskeleton has been implicated in endocytosis, yet few molecular links to the endocytic machinery have been established. Here we show that the mammalian F-actin–binding protein Abp1 (SH3P7/HIP-55) can functionally link the actin cytoskeleton to dynamin, a GTPase that functions in endocytosis. Abp1 binds directly to dynamin in vitro through its SH3 domain. Coimmunoprecipitation and colocalization studies demonstrated the in vivo relevance of this interaction. In neurons, mammalian Abp1 and dynamin colocalized at actin-rich sites proximal to the cell body during synaptogenesis. In fibroblasts, mAbp1 appeared at dynamin-rich sites of endocytosis upon growth factor stimulation. To test whether Abp1 functions in endocytosis, we overexpressed several Abp1 constructs in Cos-7 cells and assayed receptor-mediated endocytosis. While overexpression of Abp1's actin-binding modules did not interfere with endocytosis, overexpression of the SH3 domain led to a potent block of transferrin uptake. This implicates the Abp1/dynamin interaction in endocytic function. The endocytosis block was rescued by cooverexpression of dynamin. Since the addition of the actin-binding modules of Abp1 to the SH3 domain construct also fully restored endocytosis, Abp1 may support endocytosis by combining its SH3 domain interactions with cytoskeletal functions in response to signaling cascades converging on this linker protein.

scholarly journals Actin organization, bristle morphology, and viability are affected by actin capping protein mutations in Drosophila.

1996 ◽  
Vol 133 (6) ◽  
pp. 1293-1305 ◽  
Author(s):  
R Hopmann ◽  
J A Cooper ◽  
K G Miller
Keyword(s):  
Binding Proteins ◽  
Beta Subunit ◽  
Actin Binding ◽  
Filament Length ◽  
Actin Binding Proteins ◽  
Actin Organization ◽  
Capping Protein ◽  
Actin Capping Protein

Regulation of actin filament length and orientation is important in many actin-based cellular processes. This regulation is postulated to occur through the action of actin-binding proteins. Many actin-binding proteins that modify actin in vitro have been identified, but in many cases, it is not known if this activity is physiologically relevant. Capping protein (CP) is an actin-binding protein that has been demonstrated to control filament length in vitro by binding to the barbed ends and preventing the addition or loss of actin monomers. To examine the in vivo role of CP, we have performed a molecular and genetic characterization of the beta subunit of capping protein from Drosophila melanogaster. We have identified mutations in the Drosophila beta subunit-these are the first CP mutations in a multicellular organism, and unlike CP mutations in yeast, they are lethal, causing death during the early larval stage. Adult files that are heterozygous for a pair of weak alleles have a defect in bristle morphology that is correlated to disorganized actin bundles in developing bristles. Our data demonstrate that CP has an essential function during development, and further suggest that CP is required to regulate actin assembly during the development of specialized structures that depend on actin for their morphology.

scholarly journals Unexpected combinations of null mutations in genes encoding the actin cytoskeleton are lethal in yeast.

1993 ◽  
Vol 4 (5) ◽  
pp. 459-468 ◽  
Author(s):  
A E Adams ◽  
J A Cooper ◽  
D G Drubin
Keyword(s):  
Actin Cytoskeleton ◽  
Binding Proteins ◽  
Extreme Case ◽  
Actin Binding ◽  
Cell Physiology ◽  
Triple Mutant ◽  
Actin Binding Proteins ◽  
Actin Organization ◽  
Capping Protein

To understand the role of the actin cytoskeleton in cell physiology, and how actin-binding proteins regulate the actin cytoskeleton in vivo, we and others previously identified actin-binding proteins in Saccharomyces cerevisiae and studied the effect of null mutations in the genes for these proteins. A null mutation of the actin gene (ACT1) is lethal, but null mutations in the tropomyosin (TPM1), fimbrin (SAC6), Abp1p (ABP1), and capping protein (CAP1 and CAP2) genes have relatively mild or no effects. We have now constructed double and triple mutants lacking 2 or 3 of these actin-binding proteins, and studied the effect of the combined mutations on cell growth, morphology, and organization of the actin cytoskeleton. Double mutants lacking fimbrin and either Abp1p or capping protein show negative synthetic effects on growth, in the most extreme case resulting in lethality. All other combinations of double mutations and the triple mutant lacking tropomyosin, Abp1p, and capping protein, are viable and their phenotypes are similar to or only slightly more severe than those of the single mutants. Therefore, the synthetic phenotypes are highly specific. We confirmed this specificity by overexpression of capping protein and Abp1p in strains lacking fimbrin. Thus, while overexpression of these proteins has deleterious effects on actin organization in wild-type strains, no synthetic phenotype was observed in the absence of fimbrin. We draw two important conclusions from these results. First, since mutations in pairs of actin-binding protein genes cause inviability, the actin cytoskeleton of yeast does not contain a high degree of redundancy. Second, the lack of structural and functional homology among these genetically redundant proteins (fimbrin and capping protein or Abp1p) indicates that they regulate the actin cytoskeleton by different mechanisms. Determination of the molecular basis for this surprising conclusion will provide unique insights into the essential mechanisms that regulate the actin cytoskeleton.

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