The Actin Nucleator Cobl Is Controlled by Calcium and Calmodulin

Actin nucleation triggers the formation of new actin filaments and has the power to shape cells but requires tight control in order to bring about proper morphologies. The regulation of the members of the novel class of WASP Homology 2 (WH2) domain-based actin nucleators, however, thus far has largely remained elusive. Our study reveals signal cascades and mechanisms regulating Cordon-Bleu (Cobl). Cobl plays some, albeit not fully understood, role in early arborization of neurons and nucleates actin by a mechanism that requires a combination of all three of its actin monomer–binding WH2 domains. Our experiments reveal that Cobl is regulated by Ca²⁺ and multiple, direct associations of the Ca²⁺ sensor Calmodulin (CaM). Overexpression analyses and rescue experiments of Cobl loss-of-function phenotypes with Cobl mutants in primary neurons and in tissue slices demonstrated the importance of CaM binding for Cobl’s functions. Cobl-induced dendritic branch initiation was preceded by Ca²⁺ signals and coincided with local F-actin and CaM accumulations. CaM inhibitor studies showed that Cobl-mediated branching is strictly dependent on CaM activity. Mechanistic studies revealed that Ca²⁺/CaM modulates Cobl’s actin binding properties and furthermore promotes Cobl’s previously identified interactions with the membrane-shaping F-BAR protein syndapin I, which accumulated with Cobl at nascent dendritic protrusion sites. The findings of our study demonstrate a direct regulation of an actin nucleator by Ca²⁺/CaM and reveal that the Ca²⁺/CaM-controlled molecular mechanisms we discovered are crucial for Cobl’s cellular functions. By unveiling the means of Cobl regulation and the mechanisms, by which Ca²⁺/CaM signals directly converge on a cellular effector promoting actin filament formation, our work furthermore sheds light on how local Ca²⁺ signals steer and power branch initiation during early arborization of nerve cells—a key process in neuronal network formation.     

Actin cytoskeleton modulates calcium signaling during maturation of starfish oocytes

Before successful fertilization can occur, oocytes must undergo meiotic maturation. In starfish, this can be achieved in vitro by applying 1-methyladenine (1-MA). The immediate response to 1-MA is the fast Ca²⁺ release in the cell cortex. Here, we show that this Ca²⁺ wave always initiates in the vegetal hemisphere and propagates through the cortex, which is the space immediately under the plasma membrane. We have observed that alteration of the cortical actin cytoskeleton by latrunculin-A and jasplakinolide can potently affect the Ca²⁺ waves triggered by 1-MA. This indicates that the cortical actin cytoskeleton modulates Ca²⁺ release during meiotic maturation. The Ca²⁺ wave was inhibited by the classical antagonists of the InsP₃-linked Ca²⁺ signaling pathway, U73122 and heparin. To our surprise, however, these two inhibitors induced remarkable actin hyper-polymerization in the cell cortex, suggesting that their inhibitory effect on Ca²⁺ release may be attributed to the perturbation of the cortical actin cytoskeleton. In post-meiotic eggs, U73122 and jasplakinolide blocked the elevation of the vitelline layer by uncaged InsP₃, despite the massive release of Ca²⁺, implying that exocytosis of the cortical granules requires not only a Ca²⁺ rise, but also regulation of the cortical actin cytoskeleton. Our results suggest that the cortical actin cytoskeleton of starfish oocytes plays critical roles both in generating Ca²⁺ signals and in regulating cortical granule exocytosis.     

Localization and activity of calmodulin is involved in cell–cell adhesion of tumor cells and endothelial cells in response to hypoxic stress

Adhesion of tumor cells to endothelial cells is known to be involved in the hematogenous metastasis of cancer, which is regulated by hypoxia. Hypoxia is able to induce a significant increase in free intracellular Ca²⁺ levels in both tumor cells and endothelial cells. Here, we investigate the regulatory effects of calmodulin (CaM), an intracellular calcium mediator, on tumor cell–endothelial cell adhesion under hypoxic conditions. Hypoxia facilitates HeLa cell–ECV304 endothelial cell adhesion, and results in actin cytoskeleton rearrangement in both endothelial cells and tumor cells. Suppression of CaM activation by CaM inhibitor W-7 disrupts actin cytoskeleton organization and CaM distribution in the cell–cell contact region, and thus inhibits cell–cell adhesion. CaM inhibitor also downregulates hypoxia-induced HIF-1-dependent gene expression. These results suggest that the Ca²⁺-CaM signaling pathway might be involved in tumor cell-endothelial cell adhesion, and that co-localization of CaM and actin at cell–cell contact regions might be essential for this process under hypoxic stress. W.-G. Shen and W.-X. Peng Contributed to this paper equally     

EGF-like peptide-enhanced cell motility in Dictyostelium functions independently of the cAMP-mediated pathway and requires active Ca2+/calmodulin signaling

Current knowledge suggests that cell movement in the eukaryotic slime mold Dictyostelium discoideum is mediated by different signaling pathways involving a number of redundant components. Our previous research has identified a specific motility-enhancing function for epidermal growth factor-like (EGFL) repeats in Dictyostelium, specifically for the EGFL repeats of cyrA, a matricellular, calmodulin (CaM)-binding protein in Dictyostelium. Using mutants of cAMP signaling (carA⁻, carC⁻, gpaB⁻, gpbA⁻), the endogenous calcium (Ca²⁺) release inhibitor TMB-8, the CaM antagonist W-7, and a radial motility bioassay, we show that DdEGFL1, a synthetic peptide whose sequence is obtained from the first EGFL repeat of cyrA, functions independently of the cAMP-mediated signaling pathways to enhance cell motility through a mechanism involving Ca²⁺ signaling, CaM, and RasG. We show that DdEGFL1 increases the amounts of polymeric myosin II heavy chain and actin in the cytoskeleton by 24.1 ± 10.7% and 25.9 ± 2.1% respectively and demonstrate a link between Ca²⁺/CaM signaling and cytoskeletal dynamics. Finally, our findings suggest that carA and carC mediate a brake mechanism during chemotaxis since DdEGFL1 enhanced the movement of carA⁻/carC⁻ cells by 844 ± 136% compared to only 106 ± 6% for parental DH1 cells. Based on our data, this signaling pathway also appears to involve the G-protein β subunit, RasC, RasGEFA, and protein kinase B. Together, our research provides insight into the functionality of EGFL repeats in Dictyostelium and the signaling pathways regulating cell movement in this model organism. It also identifies several mechanistic components of DdEGFL1-enhanced cell movement, which may ultimately provide a model system for understanding EGFL repeat function in higher organisms.     

Apoplastic calmodulin promotes self-incompatibility pollen tube growth by enhancing calcium influx and reactive oxygen species concentration in Pyrus pyrifolia

Key message

This study indicated that Ca ²⁺ , ROS and actin filaments were involved with CaM in regulating pollen tube growth and providing a potential way for overcoming pear self-incompatibility.

Abstract

Calmodulin (CaM) has been associated with various physiological and developmental processes in plants, including pollen tube growth. In this study, we showed that CaM regulated the pear pollen tube growth in a concentration-dependent bi-phasic response. Using a whole-cell patch-clamp configuration, we showed that apoplastic CaM induced a hyperpolarization-activated calcium ion (Ca²⁺) current, and anti-CaM largely inhibited this type of Ca²⁺ current. Moreover, upon anti-CaM treatment, the reactive oxygen species (ROS) concentration decreased and actin filaments depolymerized in the pollen tube. Interestingly, CaM could partially rescue the inhibition of self-incompatible pear pollen tube growth. This phenotype could be mediated by CaM-enhanced pollen plasma membrane Ca²⁺ current, tip-localized ROS concentration and stabilized actin filaments. These data indicated that Ca²⁺, ROS and actin filaments were involved with CaM in regulating pollen tube growth and provide a potential way for overcoming pear self-incompatibility.     

Actin filament dynamics in the actomyosin VI complex is regulated allosterically by calcium-calmodulin light chain

The contractile and enzymatic activities of myosin VI are regulated by calcium binding to associated calmodulin (CaM) light chains. We have used transient phosphorescence anisotropy to monitor the microsecond rotational dynamics of erythrosin-iodoacetamide-labeled actin with strongly bound myosin VI (MVI) and to evaluate the effect of MVI-bound CaM light chain on actin filament dynamics. MVI binding lowers the amplitude but accelerates actin filament microsecond dynamics in a Ca²⁺- and CaM-dependent manner, as indicated from an increase in the final anisotropy and a decrease in the correlation time of transient phosphorescence anisotropy decays. MVI with bound apo-CaM or Ca²⁺-CaM weakly affects actin filament microsecond dynamics, relative to other myosins (e.g., muscle myosin II and myosin Va). CaM dissociation from bound MVI damps filament rotational dynamics (i.e., increases the torsional rigidity), such that the perturbation is comparable to that induced by other characterized myosins. Analysis of individual actin filament shape fluctuations imaged by fluorescence microscopy reveals a correlated effect on filament bending mechanics. These data support a model in which Ca²⁺-dependent CaM binding to the IQ domain of MVI is linked to an allosteric reorganization of the actin binding site(s), which alters the structural dynamics and the mechanical rigidity of actin filaments. Such modulation of filament dynamics may contribute to the Ca²⁺- and CaM-dependent regulation of myosin VI motility and ATP utilization.     

CBP1 Associates with the Dictyostelium Cytoskeleton and Is Important for Normal Cell Aggregation under Certain Developmental Conditions

In cells of the eukaryotic microorganism Dictyostelium discoideum, at least eight small, four-EF-hand Ca²⁺-binding proteins of unknown function are expressed at specific times during development. One of these proteins, calcium-binding protein 1 (CBP1), first appears just prior to cell aggregation and then is present at relatively constant levels throughout development. To determine a role for CBP1 during development, the protein was used as bait in a yeast two-hybrid screen to reveal putative CBP1-interacting proteins. Two proteins identified in this screen were the actin-binding proteins, protovillin and EF-1α. Using an in vitro binding assay, both of these proteins were found to interact with CBP1 in the absence of Ca²⁺, but the interaction of CBP1 with EF-1α was increased substantially by Ca²⁺. CBP1 was also shown by fluorescence microscopy and by binding assays to associate with the actin cytoskeleton of Dictyostelium cells during development, and these interactions were partially Ca²⁺-dependent. cbpA-null cells grew normally, but under certain developmental conditions, cell aggregation was prolonged and irregular. This defect in aggregation appeared to be related to a general reduction in cell motility rather than to a decrease in the ability of the cells to respond to the chemoattractant cAMP. Together, these results suggest that CBP1 might function to help regulate the reorganization of the Dictyostelium actin cytoskeleton during cell aggregation.     

Roles of Calmodulin and Calcium/Calmodulin-Dependent Protein Kinase in Flagellar Motility Regulation in the Coral Acropora Digitifera

In the corals Acropora spp., eggs secrete substances that induce sperm motility regulation. An elevation of intracellular pH ([pH]i) and a regulation of intracellular Ca²⁺ concentration ([Ca²⁺]) are involved in the sperm motility regulation cascade. However, the detailed molecular aspects of flagellar motility regulation have not been fully demonstrated in Acropora. In this study, we determined the presence and roles of both calmodulin (CaM) and calcium/calmodulin dependent-protein kinase (CaMK) in the sperm flagellar motility regulation of Acropora. A ⁴⁵Ca²⁺-overlay assay and an immunoblot analysis showed that sperm contain an acidic 16-kDa protein that was CaM, and an immunoblot analysis revealed the presence of CaMK in coral sperm. In addition, a specific inhibitor of CaMK, KN-93, and a CaM antagonist, W-7, inhibited sperm motility activation induced by NH₄Cl treatment. NH₄Cl treatment causes an increase in intracellular [pH]i of sperm, suggesting that CaM and CaMK are involved in sperm motility initiation caused by an increase in [pH]i. The involvement of CaM and CaMK in motility regulation in coral highlights the importance of these molecules throughout the animal kingdom.     

NMR analysis of free and lipid nanodisc anchored CEACAM1 membrane proximal peptides with Ca2+/CaM

CEACAM1, a homotypic transmembrane receptor with 12 or 72 amino acid cytosolic domain isoforms, is converted from inactive cis-dimers to active trans-dimers by calcium-calmodulin (Ca²⁺/CaM). Previously, the weak binding of Ca²⁺/CaM to the human 12 AA cytosolic domain was studied using C-terminal anchored peptides. We now show the binding of ¹⁵N labeled Phe-454 cytosolic domain peptides in solution or membrane anchored using NMR demonstrates a significant role for the lipid bilayer. Although binding is increased by the mutation Phe454Ala, this mutation was previously shown to abrogate actin binding. On the other hand, Ca²⁺/CaM binding is abrogated by phosphorylation of nearby Thr-457, a post-translation modification required for actin binding and subsequent in vitro lumen formation. Binding of Ca²⁺/CaM to a membrane proximal peptide from the long 72 AA cytosolic domain anchored to lipid nanodiscs was very weak compared to lipid free conditions, suggesting membrane specific effects between the two isoforms. NMR analysis of ¹⁵N labeled Ca²⁺/CaM with unlabeled peptides showed the C-lobe of Ca²⁺/CaM is involved in peptide interactions, and hydrophobic residues such as Met-109, Val-142 and Met-144 play important roles in binding peptide. This information was incorporated into transmembrane models of CEACAM1 binding to Ca²⁺/CaM. The lack of Ca²⁺/CaM binding to phosphorylated Thr-457, a residue we have previously shown to be phosphorylated by CaMK2D, also dependent on Ca²⁺/CaM, suggests stepwise binding of the cytosolic domain first to Ca²⁺/CaM and then to actin.     

Effects of exogenous calcium ions on tip growth,intracellular Ca2+ concentration,and actin arrays in hyphae of the fungus Saprolegnia ferax

The maintenance of growth of hyphae of Saprolegnia ferax was dependent on the presence of external Ca²⁺ and the growth rate increased with increased external Ca²⁺ up to 5 × 10⁻² m Ca²⁺. When Ca²⁺ was greater than 5 × 10⁻² m, growth rates decreased. Internal membrane-associated Ca²⁺ was localized with chlortetracycline. Internal Ca²⁺ became depleted in hyphae grown in the absence of Ca²⁺ and was increased in hyphae grown in high concentrations of Ca²⁺, showing that internal Ca²⁺ can be modulated by external Ca²⁺. However, the range of the internal change was not as great as the range of external concentration used, indicating that the hyphae are capable of regulating Ca²⁺ in the presence of a large concentration gradient. In the absence of external Ca²⁺, growth can occur for a limited time through use of internal Ca²⁺. The actin cytoskeleton was altered in hyphae grown in both high and low Ca²⁺. Hyphae grown in 10⁻³ m Ca²⁺ had more actin in their apical network and peripheral plaques of actin were further from the apex than in more slowly growing hyphae in 10⁻¹ m and 0 Ca²⁺. The tips of hyphae growing in low Ca²⁺ also had a tendency to swell, giving these hyphae irregular shapes. Ca²⁺ is known to affect cell wall rigidity and the consistency of actin gels, two factors that can be expected to affect hyphal growth. External Ca²⁺ does play a role in hyphal growth possibly directly by acting on the cell wall and indirectly by altering internal Ca²⁺, thus affecting the actin cytoskeleton and possibly other growth processes.     

Phosphorylation of Caldesmon at Sites between Residues 627 and 642 Attenuates Inhibitory Activity and Contributes to a Reduction in Ca-Calmodulin Affinity

Caldesmon is an actin- and myosin-binding protein found in smooth muscle that inhibits actin activation of myosin ATPase activity. The activity of caldesmon is controlled by phosphorylation and by binding to Ca²⁺-calmodulin. We investigated the effects of phosphorylation by p²¹-activated kinase 3 (PAK) and calmodulin on the 22 kDa C-terminal fragment of caldesmon (CaD22). We substituted the major PAK sites, Ser-672 and Ser-702, with either alanine or aspartic acid to mimic nonphosphorylated and constitutively phosphorylated states of caldesmon, respectively. The aspartic acid mutation of CaD22 weakened Ca²⁺-calmodulin binding but had no effect on inhibition of ATPase activity. Phosphorylation of the aspartic acid mutant with PAK resulted in the slow phosphorylation of Thr-627, Ser-631, Ser-635, and Ser-642. Phosphorylation at these sites weakened Ca²⁺-calmodulin binding further and reduced the inhibitory activity of CaD22 in the absence of Ca²⁺-calmodulin. Phosphorylation of these sites of the alanine mutant of CaD22 had no effect on Ca²⁺-calmodulin binding but did reduce inhibition of ATPase activity. Thus, the region between residues 627 and 642 may contribute to the overall regulation of caldesmon's activity.     

Modular architecture of Munc13/calmodulin complexes: dual regulation by Ca2+ and possible function in short‐term synaptic plasticity

Ca²⁺ signalling in neurons through calmodulin (CaM) has a prominent function in regulating synaptic vesicle trafficking, transport, and fusion. Importantly, Ca²⁺–CaM binds a conserved region in the priming proteins Munc13‐1 and ubMunc13‐2 and thus regulates synaptic neurotransmitter release in neurons in response to residual Ca²⁺ signals. We solved the structure of Ca²⁺₄–CaM in complex with the CaM‐binding domain of Munc13‐1, which features a novel 1‐5‐8‐26 CaM‐binding motif with two separated mobile structural modules, each involving a CaM domain. Photoaffinity labelling data reveal the same modular architecture in the complex with the ubMunc13‐2 isoform. The N‐module can be dissociated with EGTA to form the half‐loaded Munc13/Ca²⁺₂–CaM complex. The Ca²⁺ regulation of these Munc13 isoforms can therefore be explained by the modular nature of the Munc13/Ca²⁺–CaM interactions, where the C‐module provides a high‐affinity interaction activated at nanomolar [Ca²⁺]_i, whereas the N‐module acts as a sensor at micromolar [Ca²⁺]_i. This Ca²⁺/CaM‐binding mode of Munc13 likely constitutes a key molecular correlate of the characteristic Ca²⁺‐dependent modulation of short‐term synaptic plasticity.     

Phosphorylation of caldesmon during smooth muscle contraction and cell migration or proliferation

Summary The actin-binding protein caldesmon (CaD) exists both in smooth muscle (the heavy isoform, h-CaD) and non-muscle cells (the light isoform, l-CaD). In smooth muscles h-CaD binds to myosin and actin simultaneously and modulates the actomyosin interaction. In non-muscle cells l-CaD binds to actin and stabilizes␣the actin stress fibers; it may also mediate the interaction between actin and non-muscle myosins. Both h- and l-CaD are phosphorylated in vivo upon stimulation. The major phosphorylation sites of h-CaD when activated by phorbol ester are the Erk-specific sites, modification of which is attenuated by the MEK inhibitor PD98059. The same sites in l-CaD are also phosphorylated when cells are stimulated to migrate, whereas in dividing cells l-CaD is phosphorylated more extensively, presumably by cdc2 kinase. Both Erk and cdc2 are members of the MAPK family. Thus it appears that CaD is a downstream effector of the Ras signaling pathways. Significantly, the phosphorylatable serine residues shared by both CaD isoforms are in the C-terminal region that also contains the actin-binding sites. Biochemical and structural studies indicated that phosphorylation of CaD at the Erk sites is accompanied by a conformational change that partially dissociates CaD from actin. Such a structural change in h-CaD exposes the myosin-binding sites on the actin surface and allows actomyosin interactions in smooth muscles. In the case of non-muscle cells, the change in l-CaD weakens the stability of the actin filament and facilitates its disassembly. Indeed, the level of l-CaD modification correlates very well in a reciprocal manner with the level of actin stress fibers. Since both cell migration and cell division require dynamic remodeling of actin cytoskeleton that leads to cell shape changes, phosphorylation of CaD may therefore serve as a plausible means to regulate these processes. Thus CaD not only links the smooth muscle contractility and non-muscle motility, but also provides a common mechanism for the regulation of cell migration and cell proliferation.     

Ca signaling regulates ecmB expression,cell differentiation and slug regeneration in Dictyostelium

Ca²⁺ regulates cell differentiation and morphogenesis in a diversity of organisms and dysregulation of Ca²⁺ signal transduction pathways leads to many cellular pathologies. In Dictyostelium Ca²⁺ induces ecmB expression and stalk cell differentiation in vitro. Here we have analyzed the pattern of ecmB expression in intact and bisected slugs and the effect of agents that affect Ca²⁺ levels or antagonize calmodulin (CaM) on this expression pattern. We have shown that Ca²⁺ and CaM regulate ecmB expression and pstAB/pstB cell differentiation in vivo. Agents that increase intracellular Ca²⁺ levels increased ecmB expression and/or pstAB and pstB cell differentiation, while agents that decrease intracellular Ca²⁺ or antagonize CaM decreased it. In isolated slug tips agents that affect Ca²⁺ levels and antagonize CaM had differential effect on ecmB expression and cell differentiation in the anterior versus posterior zones. Agents that increase intracellular Ca²⁺ levels increased the number of ecmB expressing cells in the anterior region of slugs, while agents that decrease intracellular Ca²⁺ levels or antagonize CaM activity increased the number of ecmB expressing cells in the posterior. We have also demonstrated that agents that affect Ca²⁺ levels or antagonize CaM affect cells motility and regeneration of shape in isolated slug tips and backs and regeneration of tips in isolated slug backs. To our knowledge, this is the first study detailing the pattern of ecmB expression in regenerating slugs as well as the role of Ca²⁺ and CaM in the regeneration process and ecmB expression.     

Structure of human Na+/H+ exchanger NHE1 regulatory region in complex with calmodulin and Ca2+

The ubiquitous mammalian Na⁺/H⁺ exchanger NHE1 has critical functions in regulating intracellular pH, salt concentration, and cellular volume. The regulatory C-terminal domain of NHE1 is linked to the ion-translocating N-terminal membrane domain and acts as a scaffold for signaling complexes. A major interaction partner is calmodulin (CaM), which binds to two neighboring regions of NHE1 in a strongly Ca²⁺-dependent manner. Upon CaM binding, NHE1 is activated by a shift in sensitivity toward alkaline intracellular pH. Here we report the 2.23 Å crystal structure of the NHE1 CaM binding region (NHE1_CaMBR) in complex with CaM and Ca²⁺. The C- and N-lobes of CaM bind the first and second helix of NHE1_CaMBR, respectively. Both the NHE1 helices and the Ca²⁺-bound CaM are elongated, as confirmed by small angle x-ray scattering analysis. Our x-ray structure sheds new light on the molecular mechanisms of the phosphorylation-dependent regulation of NHE1 and enables us to propose a model of how Ca²⁺ regulates NHE1 activity.     

Requirement of reversible caldesmon phosphorylation at P21-activated kinase-responsive sites for lamellipodia extensions during cell migration

Caldesmon is believed to be one of the key regulators for actin dynamics and thereby cell polarity, membrane extension, and cell motility. We have shown previously that stress fiber formation and cell movement are severely impaired in the cells expressing human fibroblast caldesmon fragment defective in Ca2+/CaM binding sites. Both Ser458 and Ser489, adjacent to the Ca2+/CaM-binding sites, are phosphorylated by p21-activated kinase (PAK) in vitro. Here we report that Ser458 is phosphorylated in response to cell movement. We substituted Ser458 and Ser489 on C-terminal caldesmon (CaD39) with alanine or glutamic acid to mimic under-phosphorylated (CaD39-PAKA) or constitutively phosphorylated (CaD39-PAKE) caldesmon. In vitro, CaD39-PAKE, but not CaD39-PAKA, fails to inhibit myosin ATPase activity and exhibits reduced binding to Ca2+/CaM. When stably expressed in Chinese Hamster Ovary cells, both CaD39-PAKA and CaD39-PAKE incorporate into stress fibers and localize to the leading edge of the migrating cell. Expression of CaD39-PAKE, but not CaD39-PAKA, fails to protect stress fibers from cytochalasin depolymerization. However, both mutations inhibit cell polarization and lead to defects in membrane extension and cell migration. We conclude that phosphorylation of caldesmon by PAK is a dynamic process required to regulate actin dynamics and membrane protrusions in wound-induced cell migration.     

Actin,more than just a housekeeping protein at the scene of fertilization

Since the first demonstration of sperm entry into the fertilized eggs of Mediterranean sea urchin Paracentrotus lividus by Hertwig (1876), enormous progress and insights have been made on this topic. However, the precise molecular mechanisms underlying fertilization are largely unknown. The two most dramatic changes taking place in the zygote immediately after fertilization are: (i) a sharp increase of intracellular Ca²⁺ that initiates at the sperm interaction site and traverses the egg cytoplasm as a wave, and (ii) the concomitant dynamic rearrangement of the actin cytoskeleton. Traditionally, this has been studied most extensively in the sea urchin eggs, but another echinoderm, starfish, whose eggs are much bigger and transparent, has facilitated experimental approaches using microinjection and fluorescent imaging methodologies. Thus in starfish, it has been shown that the sperm-induced Ca²⁺ increase in the fertilized egg can be recapitulated by several Ca²⁺-evoking second messengers, namely inositol 1,4,5-trisphosphate (InsP3), cyclic ADP-ribose (cADPr) and nicotinic acid adenine dinucleotide phosphate (NAADP), which may play distinct roles in the generation and propagation of the Ca²⁺ waves. Interestingly, it has also been found that the dynamic rearrangement of the actin cytoskeleton in the fertilized eggs plays pivotal roles in guiding monospermic sperm entry and in the fine modulation of the intracellular Ca²⁺ signaling. As it is well known that Ca²⁺ regulates the structure of the actin cytoskeleton, our finding that Ca²⁺ signaling can be reciprocally affected by the state of the actin cytoskeleton raises an intriguing possibility that actin and Ca²⁺ signaling may form a ‘positive feedback loop’ that accelerates the downstream events of fertilization. Perturbation of the cortical actin networks also inhibits cortical granules exocytosis. Polymerizing actin bundles also compose the ‘acrosome process,’ a tubular structure protruding from the head of fertilizing sperm. Hence, actin, which is one of the most strictly conserved proteins in eukaryotes, modulates almost all major aspects of fertilization.     

Effects of genistein on β‐catenin signaling and subcellular distribution of actin‐binding proteins in human umbilical CD105‐positive stromal cells

This study was performed to define the roles of actin‐binding proteins in the regulation of actin filament assembly associated with cellular signal transduction pathways in stromal cell proliferation. Genistein, a tyrosine protein kinase inhibitor, decreased the intracellular Ca²⁺ and attenuated cell proliferation and DNA synthesis through the β‐catenin and cyclin D1 pathway in human umbilical CD105‐positive cells. Immunoprecipitation studies using anti‐β‐actin antibody revealed that several actin‐binding proteins implicated in cells include formin‐2 (FMN‐2), caldesmon (CaD), tropomyosin (Tm), and profilin. Protein levels of these proteins in whole cell lysates were not significantly changed by genistein. Three Tm isoforms, Tm‐1, Tm‐2, and Tm‐4, were found to be present in cells. Genistein caused a reduction in levels of mRNAs coding for Tm‐1 and Tm‐4, but had no significant effect on Tm‐2 mRNA levels. Immunofluorescence confocal scanning microscopy indicated that changes in the subcellular distribution of Tm and CaD, in which the diffuse cytosolic staining was shifted to show colocalization with actin stress fibers. In contrast, genistein‐induced accumulation of FMN‐2 and profilin in the peri‐nuclear area. Silencing of FMN‐2 by small interfering RNA resulted in increases of intracellular Ca²⁺ and rendered genistein resistance in decreasing intracellular Ca²⁺ in cells. These results provide the novel findings that genistein acts by modulating the cellular distribution of actin‐binding proteins in association with alterations of cellular signal transduction pathways in human stromal cell proliferation. J. Cell. Physiol. 223: 423–434, 2010. © 2010 Wiley‐Liss, Inc.     

Cytosolic Calmodulin Is Increased in SK-N-SH Human Neuroblastoma Cells Due to Release of Calcium from Intracellular Stores

Abstract: Muscarinic receptor stimulation elicits a redistribution of calmodulin (CaM) from the membrane fraction to cytosol in the human neuroblastoma cell line SK-N-SH. Increasing the intracellular Ca²⁺ concentration with ionomycin also elevates cytosolic CaM. The aim of this study was to investigate the roles of extracellular and intracellular Ca²⁺ pools in the muscarinic receptor-mediated increases in cytosolic CaM in SK-N-SH cells. Stimulus-mediated changes in intracellular Ca²⁺ were monitored in fura-2-loaded cells, and CaM was measured by radioimmunoassay in the 100,000-g cytosol and membrane fractions. The influx of extracellular Ca²⁺ normally seen with carbachol treatment in SK-N-SH cells was eliminated by pretreatment with the nonspecific Ca²⁺ channel blocker Ni²⁺. Blocking the influx of extracellular Ca²⁺ had no effect on carbachol-mediated increases in cytosolic CaM (168 ± 18% of control values for carbachol treatment alone vs. 163 ± 28% for Ni²⁺ and carbachol) or decreases in membrane CaM. Similarly, removal of extracellular Ca²⁺ from the medium did not affect carbachol-mediated increases in cytosolic CaM (168 ± 26% of control). On the other hand, prevention of the carbachol-mediated increase of intracellular free Ca²⁺ by pretreatment with the cell-permeant Ca²⁺ chelator BAPTA/AM did attenuate the carbachol-mediated increase in cytosolic CaM (221 ± 37% of control without BAPTA/AM vs. 136 ± 13% with BAPTA/AM). The effect of direct entry of extracellular Ca²⁺ into the cell by K⁺ depolarization was assessed. Incubation of SK-N-SH cells with 60 mM K⁺ elicited an immediate and persistent increase in intracellular free Ca²⁺ concentration, but there was no corresponding alteration in CaM localization. On the contrary, in cells where intracellular Ca²⁺ was directly elevated by thapsigargin treatment, cytosolic CaM was elevated for at least 30 min while particulate CaM was decreased. In addition, treatment with ionomycin in the absence of extracellular Ca²⁺, which releases Ca²⁺ from intracellular stores, induced an increase in cytosolic CaM (203 ± 30% of control). The mechanism for the CaM release may involve activation of the α isozyme of protein kinase C, which was translocated from cytosol to membranes much more profoundly by thapsigargin than by K⁺ depolarization. These data demonstrate that release of Ca²⁺ from the intracellular store is important for the carbachol-mediated redistribution of CaM in human neuroblastoma SK-N-SH cells.