Cell migration is an integrated process that involves cell adhesion, protrusion and contraction. We recently used CAS (Crk-associated substrate, 130CAS)-deficient mouse embryo fibroblasts (MEFs) to examined contribution made to v-Crk to that process via its interaction with Rac1. v-Crk, the oncogene product of avian sarcoma virus CT10, directly affects membrane ruffle formation and is associated with Rac1 activation, even in the absence of CAS, a major substrate for Crk. In CAS-deficient MEFs, cell spreading and lamellipodium dynamics are delayed; moreover, Rac activation is significantly reduced and it is no longer targeted to the membrane. However, expression of v-Crk by CAS-deficient MEFs increased cell spreading and active lamellipodium protrusion and retraction. v-Crk expression appears to induce Rac1 activation and its targeting to the membrane, which directly affects membrane dynamics and, in turn, cell migration. It thus appears that v-Crk/Rac1 signaling contributes to the regulation of membrane dynamics and cell migration, and that v-Crk is an effector molecule for Rac1 activation that regulates cell motility.
v-Crk; Rac; lamellipodia dynamics; cell migration; p130CAS
The finding that SPIN90 colocalizes with epidermal growth factor (EGF) in EEA1-positive endosomes prompted us to investigate the role of SPIN90 in endocytosis of the EGF receptor (EGFR). In the present study, we demonstrated that SPIN90 participates in the early stages of endocytosis, including vesicle formation and trafficking. Stable HeLa cells with knockdown of SPIN90 displayed significantly higher levels of surface EGFR than control cells. Analysis of the abundance and cellular distribution of EGFR via electron microscopy revealed that SPIN90 knockdown cells contain residual EGFR at cell membranes and fewer EGFR-containing endosomes, both features that reflect reduced endosome formation. The delayed early endosomal targeting capacity of SPIN90 knockdown cells led to increased EGFR stability, consistent with the observed accumulation of EGFR at the membrane. Small endosome sizes and reduced endosome formation in SPIN90 knockdown cells, observed using fluorescent confocal microscopy, strongly supported the involvement of SPIN90 in endocytosis of EGFR. Overexpression of SPIN90 variants, particularly the SH3, PRD, and CC (positions 643 - 722) domains, resulted in aberrant morphology of Rab5-positive endosomes (detected as small spots located near the cell membrane) and defects in endosomal movement. These findings clearly suggest that SPIN90 participates in the formation and movement of endosomes. Consistent with this, SPIN90 knockdown enhanced cell proliferation. The delay in EGFR endocytosis effectively increased the levels of endosomal EGFR, which triggered activation of ERK1/2 and cell proliferation via upregulation of cyclin D1. Collectively, our findings suggest that SPIN90 contributes to the formation and movement of endosomal vesicles, and modulates the stability of EGFR protein, which affects cell cycle progression via regulation of the activities of downstream proteins, such as ERK1/2, after EGF stimulation.
Membrane protrusions, like lamellipodia, and cell movement are dependent on actin dynamics, which are regulated by a variety of actin-binding proteins acting cooperatively to reorganize actin filaments. Here, we provide evidence that Swiprosin-1, a newly identified actin-binding protein, modulates lamellipodial dynamics by regulating the accessibility of F-actin to cofilin. Overexpression of Swiprosin-1 increased lamellipodia formation in B16F10 melanoma cells, whereas knockdown of Swiprosin-1 inhibited EGF-induced lamellipodia formation, and led to a loss of actin stress fibers at the leading edges of cells but not in the cell cortex. Swiprosin-1 strongly facilitated the formation of entangled or clustered F-actin, which remodeled the structural organization of actin filaments making them inaccessible to cofilin. EGF-induced phosphorylation of Swiprosin-1 at Ser183, a phosphorylation site newly identified using mass spectrometry, effectively inhibited clustering of actin filaments and permitted cofilin access to F-actin, resulting in actin depolymerization. Cells overexpressing a Swiprosin-1 phosphorylation-mimicking mutant or a phosphorylation-deficient mutant exhibited irregular membrane dynamics during the protrusion and retraction cycles of lamellipodia. Taken together, these findings suggest that dynamic exchange of Swiprosin-1 phosphorylation and dephosphorylation is a novel mechanism that regulates actin dynamics by modulating the pattern of cofilin activity at the leading edges of cells.
Electronic supplementary material
The online version of this article (doi:10.1007/s00018-013-1447-5) contains supplementary material, which is available to authorized users.
Swiprosin-1; Actin filament; Cofilin; Lamellipodia
Actin plays a fundamental role in the regulation of spine morphology (both shrinkage and enlargement) upon synaptic activation. In particular, actin depolymerization is crucial for the spine shrinkage in NMDAR-mediated synaptic depression. Here, we define the role of SPIN90 phosphorylation/dephosphorylation in regulating actin depolymerization via modulation of cofilin activity. When neurons were treated with NMDA, SPIN90 was dephosphorylated by STEP61 (striatal-enriched protein tyrosine phosphatase) and translocated from the spines to the dendritic shafts. In addition, phosphorylated SPIN90 bound cofilin and then inhibited cofilin activity, suggesting that SPIN90 dephosphorylation is a prerequisite step for releasing cofilin so that cofilin can adequately sever actin filaments into monomeric form. We found that SPIN90 YE, a phosphomimetic mutant, remained in the spines after NMDAR activation where it bound cofilin, thereby effectively preventing actin depolymerization. This led to inhibition of the activity-dependent redistribution of cortactin and drebrin A, as well as of the morphological changes in the spines that underlie synaptic plasticity. These findings indicate that NMDA-induced SPIN90 dephosphorylation and translocation initiates cofilin-mediated actin dynamics and spine shrinkage within dendritic spines, thereby modulating synaptic activity.
Electronic supplementary material
The online version of this article (doi:10.1007/s00018-013-1391-4) contains supplementary material, which is available to authorized users.
Dendritic spines; Long-term depression; Spine shrinkage; Actin depolymerization
The correct rearrangement of postsynaptic components in dendritic spines is important for driving changes of spine structure and synaptic function. SPIN90 plays an essential role in many cellular processes including actin polymerization, endocytosis, growth cone formation and dendritic spine morphogenesis. Here, we demonstrate that SPIN90, which is a binding partner of PSD95 and Shank in spines, is targeted to synapses and leads to enhanced synaptic activity in neurons. We show, using in vitro and in vivo kinase assays, that SPIN90 is tyrosine phosphorylated by Src kinase. SPIN90 that was tyrosine-phosphorylated by Src was targeted to dendritic spines in cultured hippocampal neurons. Moreover, a SPIN90 phospho-deficient mutant was unable to accumulate at dendritic spines whereas SPIN90 WT and a phospho-mimicking mutant were localized at spines and bound PSD95 and Shank with increased efficiency. Consistent with these findings, hippocampal neurons that overexpressed SPIN90 WT or a phospho-mimicking mutant had enlarged spine heads, leading to enhanced postsynaptic function in terms of both amplitude and frequency. Together, our findings show that SPIN90 modulates synaptic activity in neurons as a result of its phosphorylation.
We previously reported a novel interaction between v-Crk and myosin-1c, and demonstrated that this interaction is essential for cell migration, even in the absence of p130CAS. We here demonstrate a role for Crk-myosin-1c interaction in cell adhesion and spreading. Crk-knockout (Crk‑/‑) mouse embryo fibroblasts (MEFs) exhibited significantly decreased cell spreading and reduced Rac1 activity. A stroboscopic analysis of cell dynamics during cell spreading revealed that the cell-spreading deficiency in Crk‑/‑ MEFs was due to the short protrusion/retraction distances and long persistence times of membrane extensions. The low activity of Rac1 in Crk‑/‑ MEFs, which led to delayed cell spreading in these cells, is consistent with the observed defects in membrane dynamics. Reintroduction of v-Crk into Crk‑/‑ MEFs rescued these defects, restoring cell-spreading activity and membrane dynamics to Crk+/+ MEF levels, and normalizing Rac1 activity. Knockdown of myosin-1c by introduction of small interfering RNA resulted in a delay in cell spreading and reduced Rac1 activity to low levels, suggesting that myosin-1c also plays an essential role in cell adhesion and spreading. In addition, deletion of the v-Crk SH3 domain, which interacts with the myosin-1c tail, led to defects in cell spreading. Overexpression of the GFP-myosin-1c tail domain effectively inhibited the v-Crk-myosin-1c interaction and led to a slight decrease in cell spreading and cell surface area. Collectively, these findings suggest that the v-Crk-myosin-1c interaction, which modulates membrane dynamics by regulating Rac1 activity, is crucial for cell adhesion and spreading.
v-Crk; myosin-1c; cell adhesion; cell spreading
Synaptic plasticity is an important feature of neurons essential for learning and memory. Postsynaptic organization and composition are dynamically remodeled in response to diverse synaptic inputs during synaptic plasticity. During this process, the dynamics and localization of postsynaptic proteins are also precisely regulated. NESH/Abi-3 is a member of the Abl interactor (Abi) protein family. Overexpression of NESH is associated with reduced cell motility and tumor metastasis. Strong evidence of a close relationship between NESH and the actin cytoskeleton has been documented. Although earlier studies have shown that NESH is prominently expressed in the brain, its function and characteristics are yet to be established. Data from the present investigation suggest that synaptic localization of NESH in hippocampal neurons is regulated in an F-actin-dependent manner. The dynamic fraction of NESH in the dendritic spine was analyzed using FRAP (fluorescence recovery after photobleaching). Interestingly, F-actin stabilization and disruption significantly affected the mobile fraction of NESH, possibly through altered interactions of NESH with the F-actin. In addition, NESH was synaptically targeted from the dendritic shaft to spine after induction of chemical LTP (long-term potentiation) and the translocation was dependent on F-actin. Our data collectively support the significance of the F-actin cytoskeleton in synaptic targeting of NESH as well as its dynamics.
Dendritic spines are small membranous protrusions on the neuronal dendrites that receive synaptic input from axon terminals. Despite their importance for integrating the enormous information flow in the brain, the molecular mechanisms regulating spine morphogenesis are not well understood. NESH/Abi-3 is a member of the Abl interactor (Abi) protein family, and its overexpression is known to reduce cell motility and tumor metastasis. NESH is prominently expressed in the brain, but its function there remains unknown.
NESH was strongly expressed in the hippocampus and moderately expressed in the cerebral cortex, cerebellum and striatum, where it co-localized with the postsynaptic proteins PSD95, SPIN90 and F-actin in dendritic spines. Overexpression of NESH reduced numbers of mushroom-type spines and synapse density but increased thin, filopodia-like spines and had no effect on spine density. siRNA knockdown of NESH also reduced mushroom spine numbers and inhibited synapse formation but it increased spine density. The N-terminal region of NESH co-sedimented with filamentous actin (F-actin), which is an essential component of dendritic spines, suggesting this interaction is important for the maturation of dendritic spines.
NESH is a novel F-actin binding protein that likely plays important roles in the regulation of dendritic spine morphogenesis and synapse formation.
Endocytosis controls localization-specific signal transduction via epidermal growth factor receptor (EGFR), as well as downregulation of that receptor. Extracellular matrix (ECM)-integrin coupling induces formation of macromolecular complexes that include EGFR, integrin, Src kinase and p130Cas, resulting in EGFR activation. In addition, cell adhesion to ECM increases EGFR localization at the cell surface and reduces EGFR internalization. The molecular mechanisms involved are not yet well understood.
We investigated the molecular mechanism by which p130Cas affects the endocytic regulation of EGFR. Biochemical quantification revealed that cell adhesion to fibronectin (FN) increases total EGFR levels and its phosphorylation, and that p130Cas is required for this process. Measurements of Texas Red-labeled EGF uptake and cell surface EGFR revealed that p130Cas overexpression reduces EGF-induced EGFR internalization, while p130Cas depletion enhances it. In addition, both FN-mediated cell adhesion and p130Cas overexpression reduce EGF-stimulated dynamin phosphorylation, which is necessary for EGF-induced EGFR internalization. Coimmunoprecipitation and GST pull-down assays confirmed the interaction between p130Cas and dynamin. Moreover, a SH3-domain-deleted form of p130Cas, which shows diminished binding to dynamin, inhibits dynamin phosphorylation and EGF uptake less effectively than wild-type p130Cas.
Our results show that p130Cas plays an inhibitory role in EGFR internalization via its interaction with dynamin. Given that the EGFR internalization process determines signaling density and specificity in the EGFR pathway, these findings suggest that the interaction between p130Cas and dynamin may regulate EGFR trafficking and signaling in the same manner as other endocytic regulatory proteins related to EGFR endocytosis.
Palladin and SPIN90 are widely expressed proteins, which participate in modulation of actin cytoskeleton by binding to a variety of scaffold and signaling molecules. Cytoskeletal reorganization can induced by activation of signaling pathways, including the PDGF receptor and Src tyrosine kinase pathways. In this study we have analyzed the interplay between palladin, SPIN90 and Src, and characterized the role of palladin and SPIN90 in PDGF and Src-induced cytoskeletal remodeling. We show that the SH3 domains of SPIN90 and Src directly bind palladin’s poly-proline sequence and the interaction controls intracellular targeting of SPIN90. In PDGF-treated cells, palladin and SPIN90 co-localize in actin rich membrane ruffles and lamellipodia. The effect of PDGF on the cytoskeleton is at least partly mediated by the Src kinase, since PP2, a selective Src kinase family inhibitor, blocked PDGF-induced changes. Furthermore, expression of active Src kinase resulted in coordinated translocation of both palladin and SPIN90 to membrane protrusions. Knock-down of endogenous SPIN90 did not inhibit Src-induced cytoskeletal rearrangement, whereas knock-down of palladin resulted in cytoskeletal disorganization and inhibition of remodeling. Further studies showed that palladin is tyrosine phosphorylated in cells expressing active Src indicating bidirectional interplay between palladin and Src. These results may have implications in understanding the invasive and metastatic phenotype of neoplastic cells induced by Src.
Palladin; SPIN90; Src; cytoskeleton
Reciprocal cooperative signaling by integrins and growth factor receptors at G1 phase during cell cycle progression is well documented. By contrast, little is known about the cross-talk between integrin and transforming growth factor (TGF)-β signaling. Here, we show that integrin signaling counteracts the inhibitory effects of TGF-β on cell growth and that this effect is mediated by p130Cas (Crk-associated substrate, 130 kDa). Adhesion to fibronectin or laminin reduces TGF-β–induced Smad3 phosphorylation and thus inhibits TGF-β–mediated growth arrest; loss of p130Cas abrogates these effects. Loss and gain of function studies demonstrated that, once tyrosine-phosphorylated via integrin signaling, p130Cas binds to Smad3 and reduces phosphorylation of Smad3. That in turn leads to inhibition of p15 and p21 expression and facilitation of cell cycle progression. Thus, p130Cas-mediated control of TGF-β/Smad signaling may provide an additional clue to the mechanism underlying resistance to TGF-β–induced growth inhibition.
Calreticulin (CRT), a Ca2+-binding protein
known to have many cellular functions, including regulation of
Ca2+ homoeostasis and chaperone activity, is essential for
heart and brain development during embryogenesis in mice. Here, we
report the functional characterization of Caenorhabditis
elegans calreticulin (crt-1). A
crt-1 null mutant does not result in embryonic lethality
but shows temperature-dependent reproduction defects. In C.
elegans CRT-1 is expressed in the intestine, pharynx, body-wall
muscles, head neurons, coelomocytes, and in sperm. crt-1
males exhibit reduced mating efficiency and defects late in sperm
development in addition to defects in oocyte development and/or somatic
gonad function in hermaphrodites. Furthermore, crt-1 and
itr-1 (inositol triphosphate receptor) together
are required for normal behavioral rhythms. crt-1
transcript level is elevated under stress conditions, suggesting that
CRT-1 may be important for stress-induced chaperoning function in
The engagement of integrin α7 in E63 skeletal muscle cells by laminin or anti-α7 antibodies triggered transient elevations in the intracellular free Ca2+ concentration that resulted from both inositol triphosphate-evoked Ca2+ release from intracellular stores and extracellular Ca2+ influx through voltage-gated, L-type Ca2+ channels. The extracellular domain of integrin α7 was found to associate with both ectocalreticulin and dihydropyridine receptor on the cell surface. Calreticulin appears to also associate with cytoplasmic domain of integrin α7 in a manner highly dependent on the cytosolic Ca2+ concentration. It appeared that intracellular Ca2+ release was a prerequisite for Ca2+ influx and that calreticulin associated with the integrin cytoplasmic domain mediated the coupling of between the Ca2+ release and Ca2+ influx. These findings suggest that calreticulin serves as a cytosolic activator of integrin and a signal transducer between integrins and Ca2+ channels on the cell surface.
Apoptosis causes characteristic morphological changes in cells, including membrane blebbing, cell detachment from the extracellular matrix, and loss of cell–cell contacts. We investigated the changes in focal adhesion proteins during etoposide-induced apoptosis in Rat-1 cells and found that during apoptosis, p130cas (Crk-associated substrate [Cas]) is cleaved by caspase-3. Sequence analysis showed that Cas contains 10 DXXD consensus sites preferred by caspase-3. We identified two of these sites (DVPD416G and DSPD748G) in vitro, and point mutations substituting the Asp of DVPD416G and DSPD748G with Glu blocked caspase-3-mediated cleavage. Cleavage at DVPD416G generated a 74-kDa fragment, which was in turn cleaved at DSPD748G, yielding 47- and 31-kDa fragments. Immunofluorescence microscopy revealed well-developed focal adhesion sites in control cells that dramatically declined in number in etoposide-treated cells. Cas cleavage correlated temporally with the onset of apoptosis and coincided with the loss of p125FAK (focal adhesion kinase [FAK]) from focal adhesion sites and the attenuation of Cas–paxillin interactions. Considering that Cas associates with FAK, paxillin, and other molecules involved in the integrin signaling pathway, these results suggest that caspase-mediated cleavage of Cas contributes to the disassembly of focal adhesion complexes and interrupts survival signals from the extracellular matrix.
Protein functions are often revealed by their localization to specialized cellular sites. Recent reports demonstrated that swiprosin-1 is found together with actin and actin-binding proteins in the cytoskeleton fraction of human mast cells and NK-like cells. However, direct evidence of whether swiprosin-1 regulates actin dynamics is currently lacking. We found that swiprosin-1 localizes to microvilli-like membrane protrusions and lamellipodia and exhibits actin-binding activity. Overexpression of swiprosin-1 enhanced lamellipodia formation and cell spreading. In contrast, swiprosin-1 knockdown showed reduced cell spreading and migration. Swiprosin-1 induced actin bundling in the presence of Ca2+, and deletion of the EF-hand motifs partially reduced bundling activity. Swiprosin-1 dimerized in the presence of Ca2+ via its coiled-coil domain, and a lysine (Lys)-rich region in the coiled-coil domain was essential for regulation of actin bundling. Consistent with these observations, mutations of the EF-hand motifs and coiled-coil region significantly reduced cell spreading and lamellipodia formation. We provide new evidence of how swiprosin-1 influences cytoskeleton reorganization and cell spreading.
Intracellular accumulation of polyglutamine (polyQ)-expanded Huntingtin (Htt) protein is a hallmark of Huntington’s disease (HD). This study evaluated whether activation of Sirt1 by the anti-cancer agent, β-lapachone (β-lap), induces autophagy in human neuroblastoma SH-SY5Y cells, thereby reducing intracellular levels of polyQ aggregates and their concomitant cytotoxicity. Treatment of cells with β-lap markedly diminished the cytotoxicity induced by forced expression of Htt exon 1 containing a pathogenic polyQ stretch fused to green fluorescent protein (HttEx1(97Q)-GFP). β-lap increased autophagy in SH-SY5Y cells, as evidenced by the increased formation of LC3-II and autolysosomes. Furthermore, β-lap reduced HttEx1(97Q)-GFP aggregation, which was significantly prevented by co-incubation with 3-methyladenine, an inhibitor of autophagy. β-lap increased Sirt1 activity, as shown by the increased deacetylation of the Sirt1 substrates, PARP-1 and Atg5, and the nuclear translocation of FOXO1. Both the induction of autophagy and attenuation of HttEx1(97Q)-GFP aggregation by β-lap were significantly prevented by co-incubation with sirtinol, a general sirtuin inhibitor or by co-transfection with shRNA against Sirt1. The pro-autophagic actions of β-lap were further investigated in a transgenic Caenorhabditis elegans (C. elegans) line that expressed Q67 fused to cyanine fluorescent protein (Q67). Notably, β-lap reduced the number of Q67 puncta and restored Q67-induced defects in motility, which were largely prevented by pre-treatment with RNAi against sir-2.1, the C. elegans orthologue of Sirt1. Collectively, these data suggest that β-lap induces autophagy through activation of Sirt1, which in turn leads to a reduction in polyQ aggregation and cellular toxicity. Thus, β-lap provides a novel therapeutic opportunity for the treatment of HD.
The nuclear inclusion a (NIa) protease of turnip mosaic virus (TuMV) is responsible for the processing of the viral polyprotein into functional proteins. NIa was previously shown to possess a relatively strict substrate specificity with a preference for Val-Xaa-His-Gln↓, with the scissile bond located after Gln. The presence of the same consensus sequence, Val12-His-His-Gln15, near the presumptive α-secretase cleavage site of the amyloid-β (Aβ) peptide led us to hypothesize that NIa could possess activity against Aβ.
Western blotting results showed that oligomeric as well as monomeric forms of Aβ can be degraded by NIa in vitro. The specific cleavage of Aβ was further confirmed by mass spectrometry analysis. NIa was shown to exist predominantly in the cytoplasm as observed by immunofluorescence microscopy. The overexpression of NIa in B103 neuroblastoma cells resulted in a significant reduction in cell death caused by both intracellularly generated and exogenously added Aβ. Moreover, lentiviral-mediated expression of NIa in APPsw/PS1 transgenic mice significantly reduced the levels of Aβ and plaques in the brain.
These results indicate that the degradation of Aβ in the cytoplasm could be a novel strategy to control the levels of Aβ, plaque formation, and the associated cell death.