Involvement of actin in agonist-induced endocytosis of the G protein-coupled receptor for thromboxane A2: overcoming of actin disruption by arrestin-3 but not arrestin-2

The role of actin in endocytosis of G protein-coupled receptors is poorly defined. In the present study, we demonstrate that agents that depolymerize (latrunculin B and cytochalasin D) or stabilize (jasplakinolide) the actin cytoskeleton blocked agonist-induced endocytosis of the beta isoform of the thromboxane A(2) receptor (TPbeta) in HEK293 cells. This suggests that endocytosis of TPbeta requires active remodeling of the actin cytoskeleton. On the other hand, disruption of microtubules with colchicine did not affect endocytosis of the receptor. Expression of wild-type and mutant forms of the small GTPases RhoA and Cdc42 potently inhibited endocytosis of TPbeta, further indicating a role for the dynamic regulation of the actin cytoskeleton in this pathway. Agonist treatment of TPbeta in HEK293 cells resulted in the formation of actin stress fibers through Galpha(q/11) signaling. Because we previously showed that endocytosis of TPbeta is dependent on arrestins, we decided to explore the relation between arrestin-2 and -3 and actin in endocytosis of this receptor. Interestingly, we show that the inhibition of TPbeta endocytosis by the actin toxins in HEK293 cells was overcome by the overexpression of arrestin-3, but not of arrestin-2. These results indicate that the actin cytoskeleton is not essential in arrestin-3-mediated endocytosis of TPbeta. However, arrestin-3 could not promote endocytosis of the TPbetaY339A and TPbetaI343A carboxyl-terminal mutants when the actin cytoskeleton was disrupted. Our data provide new evidence that the actin cytoskeleton plays an essential role in TPbeta endocytosis. Furthermore, our work suggests the existence of actin-dependent and -independent arrestin-mediated pathways of endocytosis.     

Caveola-dependent endocytic entry of amphotropic murine leukemia virus

Early results suggested that the amphotropic murine leukemia virus (A-MLV) does not enter cells via endocytosis through clathrin-coated pits and this gammaretrovirus has therefore been anticipated to fuse directly with the plasma membrane. However, here we present data implicating a caveola-mediated endocytic entry route for A-MLV via its receptor Pit2. Caveolae belong to the cholesterol-rich microdomains characterized by resistance to nonionic detergents such as Triton X-100. Extraction of murine fibroblastic NIH 3T3 cells in cold Triton X-100 showed the presence of the A-MLV receptor Pit2 in detergent-insoluble microdomains. Using coimmunoprecipitation of cell extracts, we were able to demonstrate direct association of Pit2 with caveolin-1, the structural protein of caveolae. Other investigations revealed that A-MLV infection in contrast to vesicular stomatitis virus infection is a slow process (t(1/2) approximately 5 h), which is dependent on plasma membrane cholesterol but independent of NH4Cl treatment of cells; NH4Cl impairs entry via clathrin-coated pits. Furthermore, expression of dominant-negative caveolin-1 decreased the susceptibility to infection via Pit2 by approximately 70%. These results show that A-MLV can enter cells via a caveola-dependent entry route. Moreover, increase in A-MLV infection by treatment with okadaic acid as well as entry of fusion-defective fluorescent A-MLV virions in NIH 3T3 cells further confirmed our findings and show that A-MLV can enter mouse fibroblasts via an endocytic entry route involving caveolae. Finally, we also found colocalization of fusion-defective fluorescent A-MLV virions with caveolin-1 in NIH 3T3 cells. This is the first time substantial evidence has been presented implicating the existence of a caveola-dependent endocytic entry pathway for a retrovirus.     

Role of the actin cytoskeleton during influenza virus internalization into polarized epithelial cells

The in vivo site of influenza virus infection is a polarized epithelium, and it is well established that the virus preferentially enters from the apical surface of polarized epithelial cells; however, many of the molecular events involved during the endocytosis of the virus into polarized epithelia remain unclear. Here we examined the role of actin microfilaments and the myosin VI motor protein during influenza entry into a panel of polarized and non-polarized cells. By treatment of cells with cytochalasin D and jasplakinolide, we show that influenza virus entry into all the polarized epithelial cells tested requires actin dynamics, with a specific role for the actin cytoskeleton in the process of virus internalization from the plasma membrane. In contrast, influenza could still could efficiently enter and infect all non-polarized cells tested after disruption or stabilization of the actin cytoskeleton. To examine the role of the actin motor protein, myosin VI, we expressed a dominant-negative construct in both polarized and non-polarized cells. Influenza virus infectivity in myosin VI tail mutant-transfected cells was significantly decreased in polarized epithelial cells, but not in non-polarized cells. As a whole, our data suggest indispensable roles of a dynamic actin cytoskeleton for influenza virus entry into polarized epithelial cells, a feature not shared with non-polarized cells.     

Cellular entry of lymphocytic choriomeningitis virus

In contrast to most enveloped viruses that enter the host cell via clathrin-dependent endocytosis, the Old World arenavirus lymphocytic choriomeningitis virus (LCMV) enters cells via noncoated vesicles that deliver the virus to endosomes, where pH-dependent membrane fusion occurs. Here, we investigated the initial steps of LCMV infection. We found that the attachment of LCMV to its cellular receptor α-dystroglycan occurs rapidly and is not dependent on membrane cholesterol. However, subsequent virus internalization is sensitive to cholesterol depletion, indicating the involvement of a cholesterol-dependent pathway. We provide evidence that LCMV entry involves an endocytotic pathway that is independent of clathrin and caveolin and that does not require the GTPase dynamin. In addition, neither the structural integrity nor the dynamics of the actin cytoskeleton are required for infection. These findings indicate that the prototypic Old World arenavirus LCMV uses a mechanism of entry that is different from clathrin-mediated endocytosis, which is used by the New World arenavirus Junin virus, and pathways used by other enveloped viruses.     

Infectious entry of West Nile virus occurs through a clathrin-mediated endocytic pathway

The pathway of West Nile flavivirus early internalization events was mapped in detail in this study. Overexpression of dominant-negative mutants of Eps15 strongly inhibits West Nile virus (WNV) internalization, and pharmacological drugs that blocks clathrin also caused a marked reduction in virus entry but not caveola-dependent endocytosis inhibitory agent, filipin. Using immunocryoelectron microscopy, WNV particles were seen within clathrin-coated pits after 2 min postinfection. Double-labeling immunofluorescence assays and immunoelectron microscopy performed with anti-WNV envelope or capsid proteins and cellular markers (EEA1 and LAMP1) revealed the trafficking pathway of internalized virus particles from early endosomes to lysosomes and finally the uncoating of the virus particles. Disruption of host cell cytoskeleton (actin filaments and microtubules) with cytochalasin D and nocodazole showed significant reduction in virus infectivity. Actin filaments are shown to be essential during the initial penetration of the virus across the plasma membrane, whereas microtubules are involved in the trafficking of internalized virus from early endosomes to lysosomes for uncoating. Cells treated with lysosomotropic agents were largely resistant to infection, indicating that a low-pH-dependent step is required for WNV infection. In situ hybridization of DNA probes specific for viral RNA demonstrated the trafficking of uncoated viral RNA genomes to the endoplasmic reticulum.     

A novel cell entry pathway for a DAF-using human enterovirus is dependent on lipid rafts

The glycosylphosphatidylinositol (GPI)-anchored complement regulatory protein decay-accelerating factor (DAF) is used by a number of enteroviruses as a receptor during infection. DAF and other GPI-anchored proteins can be found in cholesterol-rich ordered domains within the plasma membrane that are known as "lipid rafts." We have shown, by using drugs to specifically inhibit various endocytosis routes, that infection by a DAF-using strain of echovirus 11 (EV11) is dependent upon cholesterol and an intact cytoskeleton, whereas a non-DAF-using mutant derived from it was unaffected by these drugs. Using RNA transfection and virus-binding assays, we have shown that this requirement for cholesterol, the actin cytoskeleton, and the microtubule network occurs postbinding of the virus but prior to uncoating of the RNA, indicating a role during virus entry. Confocal microscopy of virus infection supported the role of cholesterol and the cytoskeleton during entry. In addition, [(35)S]methionine-labeled DAF-using EV11, but not the non-DAF-using EV11, could be copurified with lipid raft components during infection after Triton X-100 extraction. These data indicate that DAF usage by EV11 enables the virus to associate with lipid rafts and enter cells through this novel route.     

N-Linked glycosylation is required for XC cell-specific syncytium formation by the R peptide-containing envelope protein of ecotropic murine leukemia viruses

The XC cell line undergoes extensive syncytium formation after infection with ecotropic murine leukemia viruses (MLVs) and is frequently used to titrate these viruses. This cell line is unique in its response to the ecotropic MLV envelope protein (Env) in that it undergoes syncytium formation with cells expressing Env protein containing R peptide (R(+) Env), which is known to suppress the fusogenic potential of the Env protein in other susceptible cells. To analyze the ecotropic receptor, CAT1, in XC cells, a mouse CAT1 tagged with the influenza virus hemagglutinin epitope (mCAT1-HA)-expressing retroviral vector was inoculated into XC and NIH 3T3 cells. The molecular size of the mCAT1-HA protein expressed in XC cells was smaller than that in NIH 3T3 cells due to altered N glycosylation in XC cells. Treatment of XC cells with tunicamycin significantly suppressed the formation of XC cell syncytia induced by the R(+) Env protein but not that induced by the R(-) Env protein. This result indicates that N glycosylation is required for XC cell-specific syncytium formation by the R(+) Env protein. The R(+) Env protein induced syncytia in XC cells expressing a mutant mCAT1 lacking both of two N glycosylation sites, and tunicamycin treatment suppressed syncytium formation by R(+) Env in those cells. This suggests that N glycosylation of a molecule(s) other than the receptor is required for the induction of XC cell syncytia by the R(+) Env protein.     

Matrix fibronectin binds gammaretrovirus and assists in entry: new light on viral infections

A major entry route for the gammaretrovirus amphotropic murine leukemia virus (A-MLV) into NIH 3T3 fibroblasts is via caveola-dependent endocytosis. However, during the infection time, few viral particles can be observed intracellularly. Analyzing the dynamics of the A-MLV infection process by using total internal reflection fluorescence microscopy, we show that the majority of viruses are extracellular and bound to the fibronectin matrix. Moreover, the amounts of bound virus and of fibronectin correlated. Using confocal microscopy, nanoparticles targeted to fibronectin by a III1C-fibronectin fragment or anti-fibronectin antibody were detected intracellularly in NIH 3T3 cells; unconjugated nanoparticles neither bound to cells nor were detectable intracellularly. Furthermore, A-MLV colocalized intracellularly with the fibronectin-targeted nanoparticles, suggesting that they were taken up by the same cellular pathway. Both A-MLV entry and fibronectin turnover depend on caveolar endocytosis, and we found that inhibiting viral binding to the extracellular NIH 3T3 fibronectin-matrix dramatically reduced A-MLV infection, indeed, showing an active role of fibronectin in infection. We suggest that binding to the cellular fibronectin matrix provides a new mechanism by which viruses can enter cells.     

Effects of endocytosis inhibitory drugs on rubella virus entry into VeroE6 cells

It has been suggested that infectious entry of rubella virus (RV) is conducted by receptor mediated endocytosis. To explore the cellular entry mechanism of RV, inhibitory effects of drugs affecting various endocytic pathways on RV entry into VeroE6 cells were analyzed. Results showed that RV infectious entry into VeroE6 cells is mediated by clathrin-dependent endocytosis and not by caveolae-mediated endocytosis. Moreover, chemical inhibition of macropinocytosis such as treatments of amiloride, actin and microtubule-disrupting drug significantly reduced RV infection. Considering that macropinocytosis is inducible endocytosis by cellular stimulations, clathrin-mediated endocytosis is likely to be a major route of RV infectious entry.     

Disruption of the actin cytoskeleton can complement the ability of Nef to enhance human immunodeficiency virus type 1 infectivity

The human immunodeficiency virus (HIV) protein Nef has been shown to increase the infectivity of HIV at an early point during infection. Since Nef is known to interact with proteins involved in actin cytoskeleton rearrangements, we tested the possibility that Nef may enhance HIV infectivity via a mechanism that involves the actin cytoskeleton. We find that disruption of the actin cytoskeleton complements the Nef infectivity defect. The ability of disruption of the actin cytoskeleton to complement the Nef defect was specific to envelopes that fuse at the cell surface, including a variety of HIV envelopes and the murine leukemia virus amphotropic envelope. In contrast, the infectivity of HIV virions pseudotyped to enter cells via endocytosis, which is known to complement the HIV Nef infectivity defect and can naturally penetrate the cortical actin barrier, was not altered by actin cytoskeleton disruption. The results presented here suggest that Nef functions to allow the HIV genome to penetrate the cortical actin network, a known barrier for intracellular parasitic organisms.     

Involvement of cytoskeletal components in BK virus infectious entry

Posttransplant reactivation of BK virus (BKV) in the renal allograft progresses to polyomavirus-associated nephropathy in 1% to 8% of kidney recipients. Graft dysfunction and loss in 30% to 45% of polyomavirus-associated nephropathy-affected patients are secondary to extensive tubular epithelial cell injury induced by the lytic replication of BKV. The early events in productive BKV infection are not thoroughly understood. We have previously shown that BKV enters cells by caveola-mediated endocytosis. In this report we examine the role of microfilaments and microtubules during early viral infection. Our results show that BKV infection of Vero cells is sensitive to nocodazole-induced disassembly of the microtubule network for the initial 8 hours following virus binding. In contrast, suppression of microtubule turnover with the stabilizing agent paclitaxel has no effect on BKV infectivity. Selective disassembly of the actin filaments with latrunculin A does not impede BKV infection, while inhibition of microfilament dynamics with jasplakinolide results in reduced numbers of viral antigen-positive cells. These data demonstrate that BKV, like other polyomaviruses, relies on an intact microtubule network during early infection. BKV, however, does not share the requirement with the closely related JC virus for an intact actin cytoskeleton during intracellular transport.     

ArfGAP1 restricts Mycobacterium tuberculosis entry by controlling the actin cytoskeleton

The interaction of Mycobacterium tuberculosis (Mtb) with pulmonary epithelial cells is critical for early stages of bacillus colonization and during the progression of tuberculosis. Entry of Mtb into epithelial cells has been shown to depend on F‐actin polymerization, though the molecular mechanisms are still unclear. Here, we demonstrate that mycobacterial uptake into epithelial cells requires rearrangements of the actin cytoskeleton, which are regulated by ADP‐ribosylation factor 1 (Arf1) and phospholipase D1 (PLD1), and is dependent on the M3 muscarinic receptor (M₃R). We show that this pathway is controlled by Arf GTPase‐activating protein 1 (ArfGAP1), as its silencing has an impact on actin cytoskeleton reorganization leading to uncontrolled uptake and replication of Mtb. Furthermore, we provide evidence that this pathway is critical for mycobacterial entry, while the cellular infection with other pathogens, such as Shigella flexneri and Yersinia pseudotuberculosis, is not affected. Altogether, these results reveal how cortical actin plays the role of a barrier to prevent mycobacterial entry into epithelial cells and indicate a novel role for ArfGAP1 as a restriction factor of host–pathogen interactions.     

Wild mouse variants of envelope genes of xenotropic/polytropic mouse gammaretroviruses and their XPR1 receptors elucidate receptor determinants of virus entry

Mouse xenotropic and polytropic leukemia viruses (XMVs and PMVs) are closely related gammaretroviruses that use the XPR1 receptor for entry. To identify amino acid residues in XPR1 important for virus entry, we tested mouse cells derived from evolutionarily divergent species for susceptibility to prototypical PMVs, XMVs, and the wild mouse isolate CasE#1. CasE#1 has a variant XMV/PMV host range, and sequence analysis of the CasE#1 env gene identifies segments related to PMVs and XMVs. Cells from the Asian mouse species Mus pahari show a unique pattern of susceptibility to these three viruses; these cells are susceptible to XMVs and CasE#1 but are resistant to PMVs, whereas NIH 3T3 cells show the reciprocal pattern, susceptibility to only PMVs. The M. pahari XPR1 gene differs from that of NIH 3T3 in the two extracellular loops (ECLs) previously shown to mediate virus entry (M. Marin, C. S. Tailor, A. Nouri, S. L. Kozak, and D. Kabat, J. Virol. 73:9362-9368, 1999, and N. S. Van Hoeven and A. D. Miller, Retrovirology 2:76, 2005). Using transfected hamster cells expressing chimeric and mutated XPR1s, we demonstrated that the susceptibility differences between NIH 3T3 and M. pahari cells are receptor mediated, that PMV entry requires residues in ECL3, that the CasE#1 entry determinant is in ECL4, and that determinants for XMV entry are in both ECL3 and ECL4. Additional substitutions in ECL3 and ECL4 modulate virus susceptibility and suggest that ECL3 and ECL4 may contribute to the formation of a single virus receptor site. The position of M. pahari at the base of the Mus phylogenetic tree indicates that XPR1-mediated susceptibility to XMVs is the ancestral type in this genus and that the phenotypic variants of mouse XPR1 likely arose in conjunction with exposure to gammaretrovirus infections and coevolutionary adaptations in the viral envelope.     

Involvement of cellular proteins in Junin arenavirus entry

Junin arenavirus (JUNV) entry is dependent on clathrin-mediated pathways and it relies on the integrity of the actin cytoskeleton as well as the dynamics of microtubules. To determine the method of entry used by this human pathogen, we have demonstrated that in Vero cells JUNV is trafficked via the cellular dynamin 2 (dyn2) endocytic pathway and it is dependent on the Eps15 GTPase. In addition, we have shown that the virus travels through Rab5-mediated early and Rab7-mediated late endosomes in its pH-dependent entry. Altogether, this study gives further inside into the endocytic pathway utilized by the arenavirus JUNV     

Role of protein kinase C betaII in influenza virus entry via late endosomes

Many viruses take advantage of receptor-mediated endocytosis in order to enter target cells. We have utilized influenza virus and Semliki Forest virus (SFV) to define a role for protein kinase C betaII (PKCbetaII) in endocytic trafficking. We show that specific PKC inhibitors prevent influenza virus infection, suggesting a role for classical isoforms of PKC. We also examined virus entry in cells overexpressing dominant-negative forms of PKCalpha and -beta. Cells expressing a phosphorylation-deficient form of PKCbetaII (T500V), but not an equivalent mutant form of PKCalpha, inhibited successful influenza virus entry-with the virus accumulating in late endosomes. SFV, however, believed to enter cells from the early endosome, was unaffected by PKCbetaII T500V expression. We also examined the trafficking of two cellular ligands, transferrin and epidermal growth factor (EGF). PKCbetaII T500V expression specifically blocked EGF receptor trafficking and degradation, without affecting transferrin receptor recycling. As with influenza virus, in PKCbetaII kinase-dead cells, EGF receptor was trapped in a late endosome compartment. Our findings suggest that PKCbetaII is an important regulator of a late endosomal sorting event needed for influenza virus entry and infection.     

Hepatitis B virus entry into HepG2‐NTCP cells requires clathrin‐mediated endocytosis

Hepatitis B virus (HBV) is a leading cause of cirrhosis and hepatocellular carcinoma worldwide, with 250 million individuals chronically infected. Many stages of the HBV infectious cycle have been elucidated, but the mechanisms of HBV entry remain poorly understood. The identification of the sodium taurocholate cotransporting polypeptide (NTCP) as an HBV receptor and the establishment of NTCP‐overexpressing hepatoma cell lines susceptible to HBV infection opens up new possibilities for investigating these mechanisms. We used HepG2‐NTCP cells, and various chemical inhibitors and RNA interference (RNAi) approaches to investigate the host cell factors involved in HBV entry. We found that HBV uptake into these cells was dependent on the actin cytoskeleton and did not involve macropinocytosis or caveolae‐mediated endocytosis. Instead, entry occurred via the clathrin‐mediated endocytosis pathway. HBV internalisation was inhibited by pitstop‐2 treatment and RNA‐mediated silencing (siRNA) of the clathrin heavy chain, adaptor protein AP‐2 and dynamin‐2. We were able to visualise HBV entry in clathrin‐coated pits and vesicles by electron microscopy (EM) and cryo‐EM with immunogold labelling. These data demonstrating that HBV uses a clathrin‐mediated endocytosis pathway to enter HepG2‐NTCP cells increase our understanding of the complete HBV life cycle.     

Human SCARB2-mediated entry and endocytosis of EV71

Enterovirus (EV) 71 infection is known to cause hand-foot-and-mouth disease (HFMD) and in severe cases, induces neurological disorders culminating in fatality. An outbreak of EV71 in South East Asia in 1997 affected over 120,000 people and caused neurological disorders in a few individuals. The control of EV71 infection through public health interventions remains minimal and treatments are only symptomatic. Recently, human scavenger receptor class B, member 2 (SCARB2) has been reported to be a cellular receptor of EV71. We expressed human SCARB2 gene in NIH3T3 cells (3T3-SCARB2) to study the mechanisms of EV71 entry and infection. We demonstrated that human SCARB2 serves as a cellular receptor for EV71 entry. Disruption of expression of SCARB2 using siRNAs can interfere EV71 infection and subsequent inhibit the expression of viral capsid proteins in RD and 3T3-SCARB2 but not Vero cells. SiRNAs specific to clathrin or dynamin or chemical inhibitor of clathrin-mediated endocytosis were all capable of interfering with the entry of EV71 into 3T3-SCARB2 cells. On the other hand, caveolin specific siRNA or inhibitors of caveolae-mediated endocytosis had no effect, confirming that only clathrin-mediated pathway was involved in EV71 infection. Endocytosis of EV71 was also found to be pH-dependent requiring endosomal acidification and also required intact membrane cholesterol. In summary, the mechanism of EV71 entry through SCARB2 as the receptor for attachment, and its cellular entry is through a clathrin-mediated and pH-dependent endocytic pathway. This study on the receptor and endocytic mechanisms of EV71 infection is useful for the development of effective medications and prophylactic treatment against the enterovirus.     

The requirements for viral entry differ from those for virally induced syncytium formation in NIH 3T3/DTras cells exposed to Moloney murine leukemia virus.

Moloney murine leukemia virus (Mo-MuLV) has the unique ability to infect different cells via either a low-pH-dependent or a pH-independent entry pathway. Only the pH-independent mechanism of Mo-MuLV entry has been associated with Mo-MuLV-induced syncytium formation. We have now identified a transformed cell line (NIH 3T3/DTras) which efficiently forms syncytia when exposed to Mo-MuLV, yet is low pH dependent for Mo-MuLV entry. Treatment of NIH 3T3/DTras cells with chloroquine, an agent which raises endosomal pH, blocks Mo-MuLV entry, but not Mo-MuLV-induced syncytium formation. This demonstrates that fusion which accompanies viral entry and fusion which is responsible for syncytium formation occur as independent processes in these cells. In addition, we determined that neither inherent differences in the Mo-MuLV receptor nor reduced affinity for Mo-MuLV gp70 can account for resistance of NIH 3T3 cells to Mo-MuLV-induced syncytium formation.     

Phosphoinositide-3 kinase-Akt pathway controls cellular entry of Ebola virus

The phosphoinositide-3 kinase (PI3K) pathway regulates diverse cellular activities related to cell growth, migration, survival, and vesicular trafficking. It is known that Ebola virus requires endocytosis to establish an infection. However, the cellular signals that mediate this uptake were unknown for Ebola virus as well as many other viruses. Here, the involvement of PI3K in Ebola virus entry was studied. A novel and critical role of the PI3K signaling pathway was demonstrated in cell entry of Zaire Ebola virus (ZEBOV). Inhibitors of PI3K and Akt significantly reduced infection by ZEBOV at an early step during the replication cycle. Furthermore, phosphorylation of Akt-1 was induced shortly after exposure of cells to radiation-inactivated ZEBOV, indicating that the virus actively induces the PI3K pathway and that replication was not required for this induction. Subsequent use of pseudotyped Ebola virus and/or Ebola virus-like particles, in a novel virus entry assay, provided evidence that activity of PI3K/Akt is required at the virus entry step. Class 1A PI3Ks appear to play a predominant role in regulating ZEBOV entry, and Rac1 is a key downstream effector in this regulatory cascade. Confocal imaging of fluorescently labeled ZEBOV indicated that inhibition of PI3K, Akt, or Rac1 disrupted normal uptake of virus particles into cells and resulted in aberrant accumulation of virus into a cytosolic compartment that was non-permissive for membrane fusion. We conclude that PI3K-mediated signaling plays an important role in regulating vesicular trafficking of ZEBOV necessary for cell entry. Disruption of this signaling leads to inappropriate trafficking within the cell and a block in steps leading to membrane fusion. These findings extend our current understanding of Ebola virus entry mechanism and may help in devising useful new strategies for treatment of Ebola virus infection.     

Effects of actin cytoskeleton rearrangements on channel activation by calcium entry into A431 cells

Using patch clamp and ion-selective fluorescence dye techniques, we investigated the influence of actin cytoskeleton rearrangements on the activity of calcium entry channels in plasma membrane of human carcinoma A431 cells. It is shown that disruption of actin microfilaments by cytohalasin D has no significant effect on calcium release from the stores and its entry from the extracellular space. It also does not interfere with the activation of inositol 1,4,5-trisphosphate (IP3)-dependent high-selective low-conductance calcium channels Imin. The treatment of cells with calyculin A induces the formation of actin filament layer beneath plasma membrane and also inhibits Imin activation and calcium entry through the plasma membrane, though calcium efflux from the stores was nearly unchanged. Thus, it is concluded that calcium signalling in A431 cells can be modulated by actin cytoskeleton rearrangements, and may be well described in terms of "conformational coupling" model.