Actin cytoskeleton disassembly affects conductive properties of stretch-activated cation channels in leukaemia cells

Mechanosensitive channels in various eucaryotic cells are thought to be functionally and structurally coupled to the cortical cytoskeleton. However, the results of electrophysiological studies are rather controversial and the functional impact of cytoskeleton assembly-disassembly on stretch-activated channel properties remains unclear. Here, the possible involvement of cytoskeletal elements in the regulation of stretch-activated Ca²⁺-permeable channels was studied in human leukaemia K562 cells with the use of agents that selectively modify the actin or tubulin system. F-actin disassembly resulted in a considerable reduction of the amplitude of stretch-activated currents without significant change in channel open probability. The effects of treatments with cytochalasins or latrunculin were principally similar, developed gradually and consisted a strong decrease of single channel conductance. Microtubule disruption did not affect stretch-activated channels. The data presented here are in principal agreement with the general conclusion that mechanosensitive channel functions are largely dependent on the integrity of the cortical actin cytoskeleton. Specifically, changes in conductive properties of the pore may provide an essential mechanism of channel regulation underlying functional modulation of membrane currents. Our results allow one to speculate that microfilament organization may be an important determinant in modulating biophysical characteristics of stretch-activated cation channels in cells of blood origin.     

Cholesterol depletion-induced inhibition of stretch-activated channels is mediated via actin rearrangement

Cholesterol is a critical regulator of lipid bilayer dynamics and plasma membrane organization in eukaryotes. A variety of ion channels have been shown to be modulated by cellular cholesterol and partition into cholesterol-enriched membrane rafts. However, very little is known about functional role of membrane cholesterol in regulation of mechanically gated channels that are ubiquitously present in living cells. In our previous study, the effect of methyl-beta-cyclodextrin (MbCD), cholesterol-sequestering agent, on Ca²⁺-permeable stretch-activated cation channels (SACs) has been described. Here, cell-attached patch-clamp method was employed to search for the mechanisms of cholesterol-dependent regulation of SACs and to clarify functional contribution of lipid bilayer and submembranous cytoskeleton to channel gating. Cholesterol-depleting treatment with MbCD significantly decreased open probability of SACs whereas alpha-cyclodextrin had no effect. F-actin disassembly fully restored high level of SAC activity in cholesterol-depleted cells. Particularly, treatment with cytochalasin D or latrunculin B abrogated inhibitory effect of MbCD on stretch-activated currents. Single channel analysis and fluorescent imaging methods indicate that inhibition of SACs after cholesterol depletion is mediated via actin remodeling initiated by disruption of lipid rafts. Our data reveal a novel mechanism of channel regulation by membrane cholesterol and lipid rafts.     

Stretch-Activated Potassium Channels in Hypotonically Induced Blebs of Atrial Myocytes

Stress in the lipids of the cell membrane may be responsible for activating stretch-activated channels (SACs) in nonspecialized sensory cells such as cardiac myocytes, where they are likely to play a role in cardiac mechanoelectric feedback. We examined the influence of the mechanical microenvironment on the gating of stretch-activated potassium channels (SAKCs) in rat atrial myocytes. The goal was to examine the role of the cytoskeleton in the gating process. We recorded from blebs that have minimal cytoskeleton and cells treated with cytochalasin B (cyto-B) to disrupt filamentous actin. Histochemical and electron microscopic techniques confirmed that the bleb membrane was largely free of F-actin. Channel currents showed mechanosensitivity and potassium selectivity and were activated by low pH and arachidonic acid, similar to properties of TREK-1. Some patches showed a time-dependent decrease in current that may be adaptation or inactivation, and since this decrease appeared in control cells and blebs, it is probably not the result of adaptation in the cytoskeleton. Cyto-B treatment and blebbing caused an increase in background channel activity, suggesting a transfer of stress from actin to bilayer and then to the channel. The slope sensitivity of gating before and after cyto-B treatment was similar to that of blebs, implying the characteristic change of dimensions associated with channel gating was the same in the three mechanical environments. The mechanosensitivity of SAKCs appears to be the result of interaction with membrane lipids and not of direct involvement of the cytoskeleton.     

Sodium Absorption, Volume Control and Potassium Channels: In Tribute to a Great Biologist

It is well established, for all Na-absorbing epithelia, that an increase in the rate of transcellular Na+ absorption is accompanied by an increase in the conductance of the basolateral membrane to K+. For the case of small intestinal epithelial cells from the salamander Necturus maculosus, where the rate of transcellular Na+ absorption can be increased manyfold by the addition of sugars or amino acids to the luminal bathing solution, it appears that this parallelism between Na-K pump rate and basolateral membrane K+ conductance is closely related to volume regulation by the enterocyte. Recent studies have disclosed the presence of stretch-activated K+ channels, in a highly enriched basolateral membrane fraction isolated from these epithelial cells, whose activity is increased by an increase in vesicle volume and inhibited by a decrease in vesicle volume or ATP. The activity of this channel also appears to be regulated by the degree of organization of the cortical actin cytoskeleton; activity is increased by depolymerization of the actin cytoskeleton and decreased by repolymerization of that structure. We postulate that the inhibitory effect of ATP is related to its role in promoting the polymerization of G-actin to form F-actin. We propose that enterocyte swelling that results from the intracellular accumulation of sugars or amino acids in osmotically active forms brings about disorganization of the cortical actin cytoskeleton and activates these channels and is, at least in part, responsible for the "pump-leak parallelism" in this amphibian.     

Mechanosensitive channel activity and F-actin organization in cholesterol-depleted human leukaemia cells

This study focuses on the functional role of cellular cholesterol in the regulation of mechanosensitive cation channels activated by stretch in human leukaemia K562 cells. The patch-clamp method was employed to examine the effect of methyl-beta-cyclodextrin (MbetaCD), a synthetic cholesterol-sequestering agent, on stretch-activated single currents. We found that cholesterol-depleting treatment with MbetaCD resulted in a suppression of the activity of mechanosensitive channels without a change in the unitary conductance. The probability that the channel was open significantly decreased after treatment with MbetaCD. Fluorescent microscopy revealed F-actin reorganization, possibly involving actin assembly, after incubation of the cells with MbetaCD. We suggest that suppression of mechanosensitive channel activation in cholesterol-depleted leukaemia cells is due to F-actin rearrangement, presumably induced by lipid raft destruction. Our observations are consistent with the notion that stretch-activated cation channels in eukaryotic cells are regulated by the membrane-cytoskeleton complex rather than by tension developed purely in the lipid bilayer.     

Regulation of stretch-activated intracellular calcium transients by actin filaments.

Stretch activation of cation-permeable channels may be an important proximal sensory mechanism in mechanotransduction. As actin filaments may mediate cellular responses to changes of the mechanical properties of the substrate and regulate stretch-induced calcium transients, we examined the role of actin filaments and substrate flexibility in modulating the amplitude of stretch-activated intracellular calcium transients. Human gingival fibroblasts were subjected to mechanical stretch through integrins by magnetic force acting on collagen-coated ferric oxide beads. Intracellular calcium concentration was measured in fura-2-loaded cells by ratio fluorimetry. Cytochalasin D-treatment greatly increased (3-fold) the amplitude of stretch-activated calcium transients in well-spread cells grown on glass coverslips while phalloidin, colchicine or taxol exerted no signficant effects, indicating that actin filaments but not microtubules modulate stretch-activated calcium transients. In freshly plated cells with rounded shapes and poorly developed cortical actin filaments, stretch-induced calcium transients were of 3-fold higher amplitude than well-spread cells plated for 6-24 hrs and with well developed actin filaments. Cells plated on soft collagen-polyacrylamide gels showed round morphology but exhibited <50% of the response to stretch of well-spread cells on inflexible gels. Notably, cells on soft gels showed very heavy phalloidin staining for cortical actin filaments compared with cells on more inflexible surfaces which showed only light staining for cortical actin. While cell shape may have some effect on responsiveness to mechanical stretch, the rigidity of the cell membrane mediated by the extensive cortical actin network appears to be a central determinant in the regulation of stretch-induced calcium signals.     

Osmo-sensitive and stretch-activated calcium-permeable channels in Vicia faba guard cells are regulated by actin dynamics

In responses to a number of environmental stimuli, changes of cytoplasmic [Ca(2+)](cyt) in stomatal guard cells play important roles in regulation of stomatal movements. In this study, the osmo-sensitive and stretch-activated (SA) Ca(2+) channels in the plasma membrane of Vicia faba guard cells are identified, and their regulation by osmotic changes and actin dynamics are characterized. The identified Ca(2+) channels were activated under hypotonic conditions at both whole-cell and single-channel levels. The channels were also activated by a stretch force directly applied to the membrane patches. The channel-mediated inward currents observed under hypotonic conditions or in the presence of a stretch force were blocked by the Ca(2+) channel inhibitor Gd(3+). Disruption of actin filaments activated SA Ca(2+) channels, whereas stabilization of actin filaments blocked the channel activation induced by stretch or hypotonic treatment, indicating that actin dynamics may mediate the stretch activation of these channels. In addition, [Ca(2+)](cyt) imaging demonstrated that both the hypotonic treatment and disruption of actin filaments induced significant Ca(2+) elevation in guard cell protoplasts, which is consistent with our electrophysiological results. It is concluded that stomatal guard cells may utilize SA Ca(2+) channels as osmo sensors, by which swelling of guard cells causes elevation of [Ca(2+)](cyt) and consequently inhibits overswelling of guard cells. This SA Ca(2+) channel-mediated negative feedback mechanism may coordinate with previously hypothesized positive feedback mechanisms and regulate stomatal movement in response to environmental changes.     

Mapping fungal ion channel locations

Ion channel mapping techniques are described and the results for two fungal organisms, Saprolegnia ferax and Neurospora crassa, are presented. In these species, two channel types have been characterized, stretch-activated channels exhibiting significant calcium permeability and spontaneous channels having significant potassium permeability. Two distinct analyses of patch clamp data, analysis of channel self-clustering and association between different channel types, and localization along the hyphae, reveal significant differences between the two organisms. S. ferax maintains a tip-high gradient of both channel types which is lost after disruption of the actin cytoskeleton. There is significant self-clustering of the channels, as well as interactions between channel types. N. crassa on the other hand does not maintain tip-high gradients, and clustered distributions are observed only for the stretch-activated channels. In terms of physiological roles, evidence is quite strong that the stretch-activated channels function as a growth sensor in S. ferax, but have an unknown function in N. crassa. In both organisms, the potassium permeable channels presumably function in potassium uptake. The differences between these two organisms may be due, in part, to differences in their normal environment: aquatic versus terrestrial. Copyright 1998 Academic Press.     

Mechanical compression and hydrostatic pressure induce reversible changes in actin cytoskeletal organisation in chondrocytes in agarose

In numerous cell types, the cytoskeleton has been widely implicated in mechanotransduction pathways involving stretch-activated ion channels, integrins and deformation of intracellular organelles. Studies have also demonstrated that the cytoskeleton can undergo remodelling in response to mechanical stimuli such as tensile strain or fluid flow. In articular chondrocytes, the mechanotransduction pathways are complex, inter-related and as yet, poorly understood. Furthermore, little is known of how the chondrocyte cytoskeleton responds to physiological mechanical loading. This study utilises the well-characterised chondrocyte-agarose model and an established confocal image-analysis technique to demonstrate that both static and cyclic, compressive strain and hydrostatic pressure all induce remodelling of actin microfilaments. This remodelling was characterised by a change from a uniform to a more punctate distribution of cortical actin around the cell periphery. For some loading regimes, this remodelling was reversed over a subsequent 1h unloaded period. This reversible remodelling of actin cytoskeleton may therefore represent a mechanism through which the chondrocyte alters its mechanical properties and mechanosensitivity in response to physiological mechanical loading.     

Regulation of sodium channel activity by capping of actin filaments

Ion transport in various tissues can be regulated by the cortical actin cytoskeleton. Specifically, involvement of actin dynamics in the regulation of nonvoltage-gated sodium channels has been shown. Herein, inside-out patch clamp experiments were performed to study the effect of the heterodimeric actin capping protein CapZ on sodium channel regulation in leukemia K562 cells. The channels were activated by cytochalasin-induced disruption of actin filaments and inactivated by G-actin under ionic conditions promoting rapid actin polymerization. CapZ had no direct effect on channel activity. However, being added together with G-actin, CapZ prevented actin-induced channel inactivation, and this effect occurred at CapZ/actin molar ratios from 1:5 to 1:100. When actin was allowed to polymerize at the plasma membrane to induce partial channel inactivation, subsequent addition of CapZ restored the channel activity. These results can be explained by CapZ-induced inhibition of further assembly of actin filaments at the plasma membrane due to the modification of actin dynamics by CapZ. No effect on the channel activity was observed in response to F-actin, confirming that the mechanism of channel inactivation does not involve interaction of the channel with preformed filaments. Our data show that actin-capping protein can participate in the cytoskeleton-associated regulation of sodium transport in nonexcitable cells.     

Expression of the unconventional myosin Myo1c alters sodium transport in M1 collecting duct cells

Epithelial cells rely on proper targeting of cellular components to perform their physiological function. This dynamic process utilizes the cytoskeleton and involves movement of vesicles to and from the plasma membrane, thus traversing the actin cortical cytoskeleton. Studies support both direct interaction of actin with channels and an indirect mechanism whereby actin may serve as a track in the final delivery of the channel to the plasma membrane. Actin-dependent processes are often mediated via a member of the myosin family of proteins. Myosin I family members have been implicated in multiple cellular events occurring at the plasma membrane. In these studies, we investigated the function of the unconventional myosin I Myo1c in the M1 mouse collecting duct cell line. Myo1c was observed to be concentrated at or near the plasma membrane, often in discrete membrane domains. To address the possible role of Myo1c in channel regulation, we expressed a truncated Myo1c, lacking ATP and actin domains, in M1 cells and compared electrophysiological responses to control M1 cells, M1 cells expressing the empty vector, and M1 cells expressing the full-length Myo1c construct. Interestingly, cells expressing the Myo1c constructs had modulated antidiuretic hormone (ADH)-stimulated short-circuit current and showed little inhibition of short-circuit current with amiloride addition. Evaluation of enhanced green fluorescent protein-Myo1c constructs supports the importance of the IQ region in targeting the Myo1c to its respective cellular domain. These data are consistent with Myo1c participating in the regulation of the Na⁺ channel after ADH stimulation. actin; cytoskeleton; ion channel; kidney     

Functional properties of sodium channels in cholesterol-depleted K562 cells

In the present paper, functional properties of nonvoltage-gated sodium channels in K562 cells were studied after cholesterol depletion, i.e., under conditions of the destruction of microdomains (rafts). For cholesterol depletion, cells were incubated with methyl-beta-cyclodextrin (MbCD), an oligosaccharide that selectively binds sterols. Single currents through sodium channels were recorded in cell-attached and inside-out experiments using the patch-clamp technique. After incubation with MbCD (2.5 or 5 mM), the activation of sodium channels in response to cytochalasin B or D was observed in both native cells and membrane fragments. Biophysical characteristics of sodium channels in cholesterol-depleted K562 cells were close to those in control; unitary conductance was 12 pS. Inside-out experiments with the use of globular actin have indicated that filament assembly on cytoplasmic membrane side causes an inactivation of sodium channels in the modified cells. These data imply that sodium channels in K562 cells are not associated with cholesterol-rich membrane microdomains. Possible mechanisms of the interaction of the plasma membrane and the cortical cytoskeleton are discussed.     

Tyrosine phosphorylation of Kv1.2 modulates its interaction with the actin-binding protein cortactin

Tyrosine phosphorylation evokes functional changes in a variety of ion channels. Modulation of the actin cytoskeleton also affects the function of some channels. Little is known about how these avenues of ion channel regulation may interact. We report that the potassium channel Kv1.2 associates with the actin-binding protein cortactin and that the binding is modulated by tyrosine phosphorylation. Immunocytochemical and biochemical analyses show that Kv1.2 and cortactin co-localize to the cortical actin cytoskeleton at the leading edges of the cell. Binding assays using purified recombinant proteins reveal a 19-amino acid span within the carboxyl terminus of Kv1.2 that is necessary for direct cortactin binding. Phosphorylation of specific tyrosines within the C terminus of Kv1.2 attenuates that binding. In HEK293 cells, activation of the M1 muscarinic acetylcholine receptor evokes tyrosine phosphorylation-dependent suppression of Kv1.2 ionic current. We show that M1 receptor activation also reduces the interaction of cortactin with Kv1.2 and that mutant Kv1.2 channels deficient for cortactin binding exhibit strongly attenuated ionic current. These results demonstrate a dynamic, phosphorylation-dependent interaction between Kv1.2 and the actin cytoskeleton-binding protein cortactin and suggest a role for that interaction in the regulation of Kv1.2 ionic current.     

Sodium channel activity in leukemia cells is directly controlled by actin polymerization

The actin cytoskeleton has been shown to be involved in the regulation of sodium-selective channels in non-excitable cells. However, the molecular mechanisms underlying the changes in channel function remain to be defined. In the present work, inside-out patch experiments were employed to elucidate the role of submembranous actin dynamics in the control of sodium channels in human myeloid leukemia K562 cells. We found that the application of cytochalasin D to the cytoplasmic surface of membrane fragments resulted in activation of non-voltage-gated sodium channels of 12 picosiemens conductance. Similar effects could be evoked by addition of the actin-severing protein gelsolin to the bath cytosol-like solution containing 1 microm [Ca(2+)](i). The sodium channel activity induced by disassembly of submembranous microfilaments with cytochalasin D or gelsolin could be abolished by intact actin added to the bath cytosol-like solution in the presence of 1 mm MgCl(2) to induce actin polymerization. In the absence of MgCl(2), addition of intact actin did not abolish the channel activity. Moreover, the sodium currents were unaffected by heat-inactivated actin or by actin whose polymerizability was strongly reduced by cleavage with specific Escherichia coli A2 protease ECP32. Thus, the inhibitory effect of actin on channel activity was observed only under conditions promoting rapid polymerization. Taken together, our data show that sodium channels are directly controlled by dynamic assembly and disassembly of submembranous F-actin.     

Cytoskeletal and scaffolding proteins as structural and functional determinants of TRP channels

Transient receptor potential (TRP) channels are six transmembrane-spanning proteins, with variable selectivity for cations, that play a relevant role in intracellular Ca^2 + homeostasis. There is a large body of evidence that shows association of TRP channels with the actin cytoskeleton or even the microtubules and demonstrating the functional importance of this interaction for TRP channel function. Conversely, cation currents through TRP channels have also been found to modulate cytoskeleton rearrangements. The interplay between TRP channels and the cytoskeleton has been demonstrated to be essential for full activation of a variety of cellular functions. Furthermore, TRP channels have been reported to take part of macromolecular complexes including different signal transduction proteins. Scaffolding proteins play a relevant role in the association of TRP proteins with other signaling molecules into specific microdomains. Especially relevant are the roles of the Homer family members for the regulation of TRPC channel gating in mammals and INAD in the modulation of Drosophila TRP channels. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.     

Identification of human P2X1 receptor-interacting proteins reveals a role of the cytoskeleton in receptor regulation

P2X1 receptors are ATP-gated ion channels expressed by smooth muscle and blood cells. Carboxyl-terminally His-FLAG-tagged human P2X1 receptors were stably expressed in HEK293 cells and co-purified with cytoskeletal proteins including actin. Disruption of the actin cytoskeleton with cytochalasin D inhibited P2X1 receptor currents with no effect on the time course of the response or surface expression of the receptor. Stabilization of the cytoskeleton with jasplakinolide had no effect on P2X1 receptor currents but decreased receptor mobility. P2X2 receptor currents were unaffected by cytochalasin, and P2X1/2 receptor chimeras were used to identify the molecular basis of actin sensitivity. These studies showed that the intracellular amino terminus accounts for the inhibitory effects of cytoskeletal disruption similar to that shown for lipid raft/cholesterol sensitivity. Stabilization of the cytoskeleton with jasplakinolide abolished the inhibitory effects of cholesterol depletion on P2X1 receptor currents, suggesting that lipid rafts may regulate the receptor through stabilization of the cytoskeleton. These studies show that the cytoskeleton plays an important role in P2X1 receptor regulation.     

Increased focal Kv4.2 channel expression at the plasma membrane is the result of actin depolymerization

Voltage-dependent potassium channel trafficking and localization are regulated by proteins of the cytoskeleton, but the mechanisms by which these occur are still unclear. Using human embryonic kidney (HEK) cells as a heterologous expression system, we tested the role of the actin cytoskeleton in modulating the function of Kv4.2 channels. Pretreatment (>or=1 h) of HEK cells with 5 microM cytochalasin D to disrupt the actin microfilaments greatly augmented whole cell Kv4.2 currents at potentials positive to -20 mV. However, no changes in the voltage dependence of activation and inactivation of macroscopic currents were observed to account for this increase. Similarly, single channel recordings failed to reveal any significant changes in the single channel conductance, open probability, and kinetics. However, the mean patch current was increased from 0.9 +/- 0.2 pA in control to 6.7 +/- 3.0 pA in the presence of cytochalasin D. Imaging experiments revealed a clear increase in the surface expression of the channels and the appearance of "bright spot" features, suggesting that large numbers of channels were being grouped at specific sites. Our data provide clear evidence that increased numbers and altered distribution of Kv4.2 channels at the cell surface are primarily the result of reorganization of the actin cytoskeleton.     

Forskolin Regulates L-Type Calcium Channel through Interaction between Actinin 4 and β3 Subunit in Osteoblasts

Voltage-dependent L-type calcium channels that permit cellular calcium influx are essential in calcium-mediated modulation of cellular signaling. Although the regulation of voltage-dependent L-type calcium channels is linked to many factors including cAMP-dependent protein kinase A (PKA) activity and actin cytoskeleton, little is known about the detailed mechanisms underlying the regulation in osteoblasts. Our present study investigated the modulation of L-type calcium channel activities through the effects of forskolin on actin reorganization and on its functional interaction with actin binding protein actinin 4. The results showed that forskolin did not significantly affect the trafficking of pore forming α_1c subunit and its interaction with actin binding protein actinin 4, whereas it significantly increased the expression of β₃ subunit and its interaction with actinin 4 in osteoblast cells as assessed by co-immunoprecipitation, pull-down assay, and immunostaining. Further mapping showed that the ABD and EF domains of actinin 4 were interaction sites. This interaction is independent of PKA phosphorylation. Knockdown of actinin 4 significantly decreased the activities of L-type calcium channels. Our study revealed a new aspect of the mechanisms by which the forskolin activation of adenylyl cyclase - cAMP cascade regulates the L-type calcium channel in osteoblast cells, besides the PKA mediated phosphorylation of the channel subunits. These data provide insight into the important role of interconnection among adenylyl cyclase, cAMP, PKA, the actin cytoskeleton, and the channel proteins in the regulation of voltage-dependent L-type calcium channels in osteoblast cells.     

Effect of extracellular matrix on adhesion, viability, actin cytoskeleton and K+ currents of cells expressing human ether à go-go channels

Ether à go-go (EAG) potassium channels possess oncogenic properties and have gained great interest as research tools for cancer detection and therapy. Besides, EAG electrophysiological properties are regulated through the cell cycle and determined by cytoskeletal interactions. Thus, because of the pivotal role of extracellular matrix (ECM) and cytoskeleton in cancer progression, we studied the effect of ECM components on adhesion, viability, actin organization and EAG currents in wild-type CHO cells (CHO-wt) and cells expressing human EAG channels (CHO-hEAG). At short incubation times, adhesion and viability of CHO-hEAG cells grown on collagen, heparin or poly-lysine were lower than CHO-wt cells, however, only CHO-hEAG sustained growing under total serum starvation. CHO-hEAG cells grown on poly-lysine did not organize their cytoskeleton but when grown on collagen or fibronectin displayed lamellipodia and stress fibers, respectively. Interestingly, EAG expressing cells displayed special actin structures suggesting a dynamic actin cytoskeleton, such structures were not exhibited by wild-type cells. EAG current density was significantly lower in cells grown on collagen at short incubation times. Finally, we studied potential associations between hEAG channels and integrins or actin filaments by confocal microscopy. No association between beta1-integrins and hEAG channels was found, however, a very strong co-localization was observed between hEAG channels and actin filaments, supported by immunoblot experiments in which hEAG channels were found in the insoluble fraction (associated to cytoskeleton). Our results suggest ECM components as potential modulators of oncogenic human-EAG expressing cells and emphasize the relationship between potassium channels, cytoskeleton, ECM and cancer.     

Exploring the neighborhood: adhesion-coupled cell mechanosensors

Here we discuss recent studies addressing adhesion-coupled mechanosensory processes and consider their molecular nature. Are cells using stretch-activated ion channels to explore the extracellular environment surrounding them, or do they use for that purpose the submembrane protein network that interconnects integrin receptors with the actin cytoskeleton?