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The Journal of Cell Biology Oct 2000The dynamin family of large GTPases has been implicated in the formation of nascent vesicles in both the endocytic and secretory pathways. It is believed that dynamin...
The dynamin family of large GTPases has been implicated in the formation of nascent vesicles in both the endocytic and secretory pathways. It is believed that dynamin interacts with a variety of cellular proteins to constrict membranes. The actin cytoskeleton has also been implicated in altering membrane shape and form during cell migration, endocytosis, and secretion and has been postulated to work synergistically with dynamin and coat proteins in several of these important processes. We have observed that the cytoplasmic distribution of dynamin changes dramatically in fibroblasts that have been stimulated to undergo migration with a motagen/hormone. In quiescent cells, dynamin 2 (Dyn 2) associates predominantly with clathrin-coated vesicles at the plasma membrane and the Golgi apparatus. Upon treatment with PDGF to induce cell migration, dynamin becomes markedly associated with membrane ruffles and lamellipodia. Biochemical and morphological studies using antibodies and GFP-tagged dynamin demonstrate an interaction with cortactin. Cortactin is an actin-binding protein that contains a well defined SH3 domain. Using a variety of biochemical methods we demonstrate that the cortactin-SH3 domain associates with the proline-rich domain (PRD) of dynamin. Functional studies that express wild-type and mutant forms of dynamin and/or cortactin in living cells support these in vitro observations and demonstrate that an increased expression of cortactin leads to a significant recruitment of endogenous or expressed dynamin into the cell ruffle. Further, expression of a cortactin protein lacking the interactive SH3 domain (CortDeltaSH3) significantly reduces dynamin localization to the ruffle. Accordingly, transfected cells expressing Dyn 2 lacking the PRD (Dyn 2(aa)DeltaPRD) sequester little of this protein to the cortactin-rich ruffle. Interestingly, these mutant cells are viable, but display dramatic alterations in morphology. This change in shape appears to be due, in part, to a striking increase in the number of actin stress fibers. These findings provide the first demonstration that dynamin can interact with the actin cytoskeleton to regulate actin reorganization and subsequently cell shape.
Topics: Amino Acid Sequence; Binding Sites; Cell Movement; Cell Size; Cortactin; Dynamin I; Dynamins; Fluorescent Antibody Technique; GTP Phosphohydrolases; Microfilament Proteins; Molecular Sequence Data; Mutation; Protein Binding; Protein Structure, Tertiary; Pseudopodia; Sequence Deletion; src Homology Domains
PubMed: 11018064
DOI: 10.1083/jcb.151.1.187 -
Plant Physiology Aug 2008Actin microfilaments are crucial for polar cell tip growth, and their configurations and dynamics are regulated by the actions of various actin-binding proteins (ABPs)....
Actin microfilaments are crucial for polar cell tip growth, and their configurations and dynamics are regulated by the actions of various actin-binding proteins (ABPs). We explored the function of a lily (Lilium longiflorum) pollen-enriched LIM domain-containing protein, LlLIM1, in regulating the actin dynamics in elongating pollen tube. Cytological and biochemical assays verified LlLIM1 functioning as an ABP, promoting filamentous actin (F-actin) bundle assembly and protecting F-actin against latrunculin B-mediated depolymerization. Overexpressed LlLIM1 significantly disturbed pollen tube growth and morphology, with multiple tubes protruding from one pollen grain and coaggregation of FM4-64-labeled vesicles and Golgi apparatuses at the subapex of the tube tip. Moderate expression of LlLIM1 induced an oscillatory formation of asterisk-shaped F-actin aggregates that oscillated with growth period but in different phases at the subapical region. These results suggest that the formation of LlLIM1-mediated overstabilized F-actin bundles interfered with endomembrane trafficking to result in growth retardation. Cosedimentation assays revealed that the binding affinity of LlLIM1 to F-actin was simultaneously regulated by both pH and Ca(2+): LlLIM1 showed a preference for F-actin binding under low pH and low Ca(2+) concentration. The potential functions of LlLIM1 as an ABP sensitive to pH and calcium in integrating endomembrane trafficking, oscillatory pH, and calcium circumstances to regulate tip-focused pollen tube growth are discussed.
Topics: Actin Cytoskeleton; Actins; Amino Acid Sequence; Calcium; Cloning, Molecular; Germination; Golgi Apparatus; Hydrogen; Hydrogen-Ion Concentration; Lilium; Microfilament Proteins; Molecular Sequence Data; Plant Proteins; Pollen Tube; Protein Isoforms; Sequence Alignment; Signal Transduction; Transport Vesicles
PubMed: 18480376
DOI: 10.1104/pp.108.118604 -
Trends in Biochemical Sciences Oct 2019Transgelin-2 has been regarded as an actin-binding protein that induces actin gelation and regulates actin cytoskeleton. However, transgelin-2 has recently been shown to... (Review)
Review
Transgelin-2 has been regarded as an actin-binding protein that induces actin gelation and regulates actin cytoskeleton. However, transgelin-2 has recently been shown to relax the myosin cytoskeleton of the airway smooth muscle cells by acting as a receptor for extracellular metallothionein-2. From a clinical perspective, these results support transgelin-2 as a promising therapeutic target for diseases such as cancer and asthma. The inhibition of transgelin-2 prevents actin gelation and thereby cancer cell proliferation, invasion, and metastasis. Conversely, the activation of transgelin-2 with specific agonists relaxes airway smooth muscles and reduces pulmonary resistance in asthma. Here, we review new studies on the biochemical properties of transgelin-2 and discuss their clinical implications for the treatment of immune, oncogenic, and respiratory disorders.
Topics: Actins; Animals; Asthma; Cell Proliferation; Humans; Microfilament Proteins; Muscle Proteins; Neoplasms
PubMed: 31256982
DOI: 10.1016/j.tibs.2019.05.004 -
BMB Reports Jan 2016Spectraplakins are crucially important communicators, linking cytoskeletal components to each other and cellular junctions. Microtubule actin crosslinking factor 1... (Review)
Review
Spectraplakins are crucially important communicators, linking cytoskeletal components to each other and cellular junctions. Microtubule actin crosslinking factor 1 (MACF1), also known as actin crosslinking family 7 (ACF7), is a member of the spectraplakin family. It is expressed in numerous tissues and cells as one extensively studied spectraplakin. MACF1 has several isoforms with unique structures and well-known function to be able to crosslink F-actin and microtubules. MACF1 is one versatile spectraplakin with various functions in cell processes, embryo development, tissue-specific functions, and human diseases. The importance of MACF1 has become more apparent in recent years. Here, we summarize the current knowledge on the presence and function of MACF1 and provide perspectives on future research of MACF1 based on our studies and others.
Topics: Actins; Animals; Cell Movement; Embryo, Mammalian; Humans; Microfilament Proteins; Protein Binding; Protein Isoforms; Protein Structure, Tertiary; Signal Transduction
PubMed: 26521939
DOI: 10.5483/BMBRep.2016.49.1.185 -
Progress in Neurobiology Mar 2021While positive regulators of hippocampal long-term potentiation (LTP) have extensively been investigated, relatively little is known about the inhibitory regulators of...
While positive regulators of hippocampal long-term potentiation (LTP) have extensively been investigated, relatively little is known about the inhibitory regulators of LTP. We previously reported that Cyclin Y (CCNY), a member of cyclin family generally known to function in proliferating cells, is a novel postsynaptic protein that serves as a negative regulator of functional LTP. However, whether CCNY plays a role in structural LTP, which is mechanistically linked to functional LTP, and which mechanisms are involved in the CCNY-mediated suppression of LTP at the molecular level remain elusive. Here, we report that CCNY negatively regulates the plasticity-induced changes in spine morphology through the control of actin dynamics. We observed that CCNY directly binds to filamentous actin and interferes with LTP-induced actin polymerization as well as depolymerization by blocking the activation of cofilin, an actin-depolymerizing factor, thus resulting in less plastic spines and the impairment of structural LTP. These data suggest that CCNY acts as an inhibitory regulator for both structural and functional LTP by modulating actin dynamics through the cofilin-actin pathway. Collectively, our findings provide a mechanistic insight into the inhibitory modulation of hippocampal LTP by CCNY, highlighting a novel function of a cyclin family protein in non-proliferating neuronal cells.
Topics: Actin Depolymerizing Factors; Actins; Cyclins; Microfilament Proteins; Neuronal Plasticity; Synapses
PubMed: 32966834
DOI: 10.1016/j.pneurobio.2020.101915 -
FEBS Letters Apr 2020Anillin is a type of actin filament cross-linking protein that stabilizes the actin-based contractile ring during cytokinesis. To elucidate the underlying intermolecular...
Anillin is a type of actin filament cross-linking protein that stabilizes the actin-based contractile ring during cytokinesis. To elucidate the underlying intermolecular interactions between actin filaments and anillin, we utilized total internal reflection fluorescence microscopy (TIRFM) and high-speed atomic force microscopy (Hs-AFM). Single-molecule imaging of anillin using TIRFM showed that anillin exists as monomers with relatively low binding affinity for actin filaments. Real-time imaging of actin filament cross-linking dynamics induced by anillin using Hs-AFM revealed that anillin monomers cross-link with actin filaments at a distance of 8 nm and that the polarity of those filaments is both parallel and antiparallel. These results are consistent with anillin playing a role in actin ring transition in vivo, where it might be responsible for thinning the ring-shaped apolar actin bundles.
Topics: Actin Cytoskeleton; Actins; Binding Sites; Green Fluorescent Proteins; HEK293 Cells; Humans; Microfilament Proteins; Microscopy, Atomic Force; Microscopy, Fluorescence; Molecular Imaging; Photobleaching
PubMed: 31853940
DOI: 10.1002/1873-3468.13720 -
PloS One 2013Insulin stimulates glucose uptake through a highly organized and complex process that involves movement of the glucose transporter 4 (GLUT4) from intracellular storage...
Insulin stimulates glucose uptake through a highly organized and complex process that involves movement of the glucose transporter 4 (GLUT4) from intracellular storage sites to the plasma membrane. Previous studies in L6 skeletal muscle cells have shown that insulin-induced activation and assembly of insulin receptor substrate 1 (IRS1) and p85α the regulatory subunit of the Type 1A phosphatidylinositol-3-kinase (PI3K), within remodeled actin-rich membrane structures is critical for downstream signalling mediating the translocation of GLUT4. The mechanism for localization within actin cytoskeletal scaffolds is not known, as direct interaction of IRS1 or p85α with F-actin has not been demonstrated. Here we show that nexilin, a F-actin binding protein implicated in the pathogenesis of familial dilated cardiomyopathies, preferentially binds to IRS1 over IRS2 to influence glucose transport in skeletal muscle cells. Nexilin stably associates with IRS1 under basal conditions in L6 myotubes and this complex is disassembled by insulin. Exposure of L6 myotubes to Latrunculin B disrupts the spatial patterning of nexilin and its transient association with IRS1. Functional silencing of nexilin has no effect on insulin-stimulated IRS1 tyrosine phosphorylation, however it enhances recruitment of p85α to IRS1 resulting in increased PI-3, 4, 5-P(3) formation, coincident with enhanced AKT activation and glucose uptake. By contrast, overexpression of nexilin inhibits transmission of IRS1 signals to AKT. Based on these findings we propose that nexilin may tether IRS1 to actin-rich structures under basal conditions, confining IRS1 signaling to specific subcellular locations in the cell. Insulin-elicited release of this constraint may enhance the efficiency of IRS1/PI3K interaction and PI-3, 4, 5-P(3) production at localized sites. Moreover, the selective binding of nexilin to IRS1 and not IRS2 may contribute to the differential specificity of IRS isoforms in the modulation of GLUT4 trafficking in skeletal muscle cells.
Topics: 3T3-L1 Cells; Actins; Animals; Cell Line; Gene Expression; Gene Silencing; Glucose; Insulin; Insulin Receptor Substrate Proteins; Mice; Microfilament Proteins; Muscle Cells; Muscle, Skeletal; Phosphatidylinositol 3-Kinases; Protein Binding; Protein Transport; Proto-Oncogene Proteins c-akt; Rats; Signal Transduction
PubMed: 23383252
DOI: 10.1371/journal.pone.0055634 -
The Journal of Biological Chemistry Nov 2013Higher order actin filament structures are necessary for cytoplasmic streaming, organelle movement, and other physiological processes. However, the mechanism by which...
Higher order actin filament structures are necessary for cytoplasmic streaming, organelle movement, and other physiological processes. However, the mechanism by which the higher order cytoskeleton is formed in plants remains unknown. In this study, we identified a novel actin-cross-linking protein family (named CROLIN) that is well conserved only in the plant kingdom. There are six isovariants of CROLIN in the Arabidopsis genome, with CROLIN1 specifically expressed in pollen. In vitro biochemical analyses showed that CROLIN1 is a novel actin-cross-linking protein with binding and stabilizing activities. Remarkably, CROLIN1 can cross-link actin bundles into actin networks. CROLIN1 loss of function induces pollen germination and pollen tube growth hypersensitive to latrunculin B. All of these results demonstrate that CROLIN1 may play an important role in stabilizing and remodeling actin filaments by binding to and cross-linking actin filaments.
Topics: Actins; Amino Acid Sequence; Arabidopsis; Arabidopsis Proteins; Microfilament Proteins; Molecular Sequence Data; Pollen Tube
PubMed: 24072702
DOI: 10.1074/jbc.M113.483594 -
The Journal of Comparative Neurology Sep 2012Filamin A (FLNa) is an actin-binding protein that regulates cell motility, adhesion, and elasticity by cross-linking filamentous actin. Additional roles of FLNa include...
Filamin A (FLNa) is an actin-binding protein that regulates cell motility, adhesion, and elasticity by cross-linking filamentous actin. Additional roles of FLNa include regulation of protein trafficking and surface expression. Although the functions of FLNa during brain development are well studied, little is known on its expression, distribution, and function in the adult brain. Here we characterize in detail the neuroanatomical distribution and subcellular localization of FLNa in the mature rat brain, by using two antisera directed against epitopes at either the N' or the C' terminus of the protein, further validated by mRNA expression. FLNa was widely and selectively expressed throughout the brain, and the intensity of immunoreactivity was region dependent. The most intensely FLNa-labeled neurons were found in discrete neuronal systems, including basal forebrain structures, anterior nuclear group of thalamus, and hypothalamic parvocellular neurons. Pyramidal neurons in neocortex and hippocampus and magnocellular cells in basolateral amygdaloid nucleus were also intensely FLNa immunoreactive, and strong FLNa labeling was evident in the pontine and medullary raphe nuclei and in sensory and spinal trigeminal nuclei. The subcellular localization of FLNa was evaluated in situ as well as in primary hippocampal neurons. Punctate expression was found in somata and along the dendritic shaft, but FLNa was not detected in dendritic spines. These subcellular distribution patterns were recapitulated in hippocampal and neocortical pyramidal neurons in vivo. The characterization of the expression and subcellular localization of FLNa may provide new clues to the functional roles of this cytoskeletal protein in the adult brain.
Topics: Animals; Blotting, Western; Brain; Contractile Proteins; Filamins; Immunohistochemistry; In Situ Hybridization; Microfilament Proteins; Neurons; Rats; Rats, Sprague-Dawley
PubMed: 22434607
DOI: 10.1002/cne.23106 -
Proceedings of the National Academy of... Jul 2022The abLIM1 is a nonerythroid actin-binding protein critical for stable plasma membrane-cortex interactions under mechanical tension. Its depletion by RNA interference...
The abLIM1 is a nonerythroid actin-binding protein critical for stable plasma membrane-cortex interactions under mechanical tension. Its depletion by RNA interference results in sparse, poorly interconnected cortical actin networks and severe blebbing of migrating cells. Its isoforms, abLIM-L, abLIM-M, and abLIM-S, contain, respectively four, three, and no LIM domains, followed by a C terminus entirely homologous to erythroid cortex protein dematin. How abLIM1 functions, however, remains unclear. Here we show that abLIM1 is a liquid-liquid phase separation (LLPS)-dependent self-organizer of actin networks. Phase-separated condensates of abLIM-S-mimicking ΔLIM or the major isoform abLIM-M nucleated, flew along, and cross-linked together actin filaments (F-actin) to produce unique aster-like radial arrays and interconnected webs of F-actin bundles. Interestingly, ΔLIM condensates facilitated actin nucleation and network formation even in the absence of Mg. Our results suggest that abLIM1 functions as an LLPS-dependent actin nucleator and cross-linker and provide insights into how LLPS-induced condensates could self-construct intracellular architectures of high connectivity and plasticity.
Topics: Actin Cytoskeleton; Actins; Humans; LIM Domain Proteins; Microfilament Proteins; Protein Isoforms; RNA Interference
PubMed: 35858327
DOI: 10.1073/pnas.2122420119