Vinculin is a permanent component of the membrane skeleton and is incorporated into the (re)organising cytoskeleton upon platelet activation

Vinculin, a 130-kDa protein discovered in chicken gizzard smooth-muscle cells and subsequently also described in platelets, is believed to be involved in membrane-cytoskeleton interactions. In this study we investigated vinculin distribution in human blood platelets. Two skeletal fractions and a remaining cytosolic fraction were prepared with a recently described Triton X-100 lysis buffer causing minimal post-lysis breakdown by proteolysis. The presence of vinculin was demonstrated in the membrane skeleton and cytosol of resting and thrombin-activated human platelets. Upon thrombin stimulation vinculin also appeared in the cytoskeleton. this cytoskeletal incorporation was completed during the early stages of platelet aggregation and secretion, when the uptake of myosin, actin-binding protein and talin was still not maximal. We conclude therefore, that vinculin may play an important role in the structural (re)organisation of the human platelet cytoskeleton upon platelet activation.     

Cytoskeleton involvement in the distribution of mRNP complexes and small cytoplasmic RNAs

These studies were designed to determine whether small cytoplasmic RNAs and two different mRNAs (actin mRNA and histone H4 mRNA) were uniformly distributed among various subcellular compartments. The cytoplasm of HeLa S3 cells was fractionated into four RNA-containing compartments. The RNAs bound to the cytoskeleton were separated from those in the soluble cytoplasmic phase and each RNA fraction was further separated into those bound and those not bound to polyribosomes. The four cytoplasmic RNA fractions were analysed to determine which RNA species were present in each. The 7 S RNAs were found in all cytoplasmic fractions, as were the 5 S and 5.8 S ribosomal RNAs, while transfer RNA was found largely in the soluble fraction devoid of polysomes. On the other hand a group of prominent small cytoplasmic RNAs (scRNAs of 105-348 nucleotides) was isolated from the fraction devoid of polysomes but bound to the cytoskeleton. Actin mRNA was found only in polyribosomes bound to the cytoskeleton. This mRNA was released into the soluble phase by cytochalasin B treatment, suggesting a dependence upon actin filament integrity for cytoskeletal binding. A significant portion of several scRNAs was also released from the cytoskeleton by cytochalasin B treatment. Analysis of the spatial distribution of histone H4 mRNAs, however, revealed a more widely dispersed message. Although most (60%) of the H4 mRNA was associated with polyribosomes in the soluble phase, a significant amount was also recovered in both of the cytoskeleton bound fractions either associated or free of polyribosome interaction. Treatment with cytochalasin B suggested that only cytoskeleton bound, untranslated H4 mRNA was dependent upon the integrity of actin filaments for cytoskeletal binding.     

Heat shock protein HSP90 and its association with the cytoskeleton: a morphological study.

To investigate the cellular localization of the 90-kilodalton heat shock protein (HSP90) and its interaction with the cytoskeleton, we performed single- and double-staining immunofluorescence microscopy of cytoskeletal proteins and HSP90 in the absence and presence of cytoskeletal inhibitors. As a model, we used a human endometrial adenocarcinoma cell line (Ishikawa cells), which expresses HSP90. We confirmed the recently reported colocalization of HSP90 with microtubules. However, Ishikawa cells treated with 10(-5) M of the antimicrotubule agents colchicine or triethyl lead showed residual filamentous structures stained with anti-HSP90 antibodies, while no microtubules were visualized with anti-tubulin antibodies. In the presence of 10(-5) M cytochalasin B, the microfilament staining of the cells disappeared, while residual filamentous structures were labeled with anti-HSP90 antibodies. Furthermore, Ishikawa cells treated with 10(-5) M triethyl lead and stained with anti-HSP90 antibodies demonstrated residual filamentous structures, clearly different from those of reorganized vimentin intermediate filaments. Conversely, similar reorganized morphology of filamentous structures stained with both anti-HSP90 and anti-cytokeratins antibodies were observed when Ishikawa cells were treated with 2 x 10(-5) M cytochalasin B and 2 x 10(-5) M colchicine. HSP90 was also present in Ishikawa cell preparations of the Triton X-100 insoluble cytoskeleton. In addition, Triton-insoluble cytoskeleton treated with 10(-5). M triethyl lead and double stained with anti-HSP90 and anti-vimentin antibodies demonstrated clearly different filamentous patterns, when exposed on the same photographic plaque.(ABSTRACT TRUNCATED AT 250 WORDS)     

Membrane tether formation from blebbing cells

Membrane tension has been proposed to be important in regulating cell functions such as endocytosis and cell motility. The apparent membrane tension has been calculated from tether forces measured with laser tweezers. Both membrane-cytoskeleton adhesion and membrane tension contribute to the tether force. Separation of the plasma membrane from the cytoskeleton occurs in membrane blebs, which could remove the membrane-cytoskeleton adhesion term. In renal epithelial cells, tether forces are significantly lower on blebs than on membranes that are supported by cytoskeleton. Furthermore, the tether forces are equal on apical and basolateral blebs. In contrast, tether forces from membranes supported by the cytoskeleton are greater in apical than in basolateral regions, which is consistent with the greater apparent cytoskeletal density in the apical region. We suggest that the tether force on blebs primarily contains only the membrane tension term and that the membrane tension may be uniform over the cell surface. Additional support for this hypothesis comes from observations of melanoma cells that spontaneously bleb. In melanoma cells, tether forces on blebs are proportional to the radius of the bleb, and as large blebs form, there are spikes in the tether force in other cell regions. We suggest that an internal osmotic pressure inflates the blebs, and the pressure calculated from the Law of Laplace is similar to independent measurements of intracellular pressures. When the membrane tension term is subtracted from the apparent membrane tension over the cytoskeleton, the membrane-cytoskeleton adhesion term can be estimated. In both cell systems, membrane-cytoskeleton adhesion was the major factor in generating the tether force.     

Differential association of membrane-bound and non-membrane-bound polysomes with the cytoskeleton

We report here a differential release of specific mRNAs from the cytoskeleton by cytochalasin D treatment. Non-membrane-bound polysomal mRNAs, such as histone mRNA and c-fos mRNA, are readily released from the cytoskeleton of HeLa cells during cytochalasin D treatment. Over 90% of H3 and H4 histone mRNA is associated with the cytoskeleton in control cells and only 25% in cells treated with cytochalasin D (40 micrograms/ml). In contrast, the membrane-bound polysomal mRNAs for HLA-B7 and chorionic gonadotropin-alpha are inefficiently released from the cytoskeletal framework by cytochalasin D alone; approximately 98% of the HLA-B7 mRNA in control cells is associated with the cytoskeleton, whereas approximately 65% of the HLA-B7 mRNA is retained on the cytoskeleton in cells treated with cytochalasin D (40 micrograms/ml). Disruption of polysome structure with puromycin during cytochalasin D treatment results in the efficient release of HLA-B7 mRNA from the cytoskeleton. Under these conditions, only 25% of the HLA-B7 mRNA remains associated with the cytoskeletal framework. Thus, membrane-bound polysomes appear to be attached to the cytoskeleton through a cytochalasin D-sensitive site as well as through association with the nascent polypeptide and/or ribosome. These results demonstrate a complex association of polysomes with the cytoskeleton and elements of the endoplasmic reticulum.     

Agorins: major structural proteins of the plasma membrane skeleton of P815 tumor cells

Plasma membranes of P815 mastocytoma cells contain a set of proteins that remain selectively insoluble upon extraction of the membranes with Triton X-100, and appear to form a membrane skeletal matrix independent of the filamentous cytoskeletal systems. EGTA treatment of the matrix was found to release approximately 25% of the protein as polypeptides of 70, 69, 38, and 36 kD, all of which appear to be peripheral components associated with the cytoplasmic face of the plasma membrane via divalent cation-dependent interactions. About 75% of the total matrix protein was recovered in the EGTA-insoluble fraction. Actin accounted for approximately 5% of the total protein in the EGTA-insoluble fraction. The rest was accounted for by two novel proteins of 20 and 40 kD which, despite their relatively low molecular weights, do not enter SDS PAGE gels. Together these proteins account for approximately 15% of the total plasma membrane protein, and are thus present in much higher amounts than any other characterized protein of nucleated cell plasma membranes. Based on the extensive associations of these proteins to form very large detergent-insoluble structures, we propose that they may be named agorin I, the 20-kD protein, and agorin II, the 40-kD protein, from the Greek agora meaning assembly. The amount and properties of these proteins and the appearance of the EGTA-insoluble material in thin-section electron micrographs indicate that the agorins are the major structural elements of the membrane matrix, and thus of the putative membrane skeleton.     

Association of the actin cytoskeleton with glass-adherent proteins in mouse peritoneal macrophages

Summary— When mouse peritoneal macrophages adherent to glass surface were removed by treatment with triethanolamine and Nonidet P-40, fine thread structures of unique loops were left behind on glass at the sites of cell adhesion. To examine the ultrastructural relationship between such looped threads and cytoskeletal components in glass-adherent macrophages, we successfully used the ‘zinc method’ to remove most of the cytoplasm including nuclei and to expose the cytoskeleton associated with the ventral plasma membrane. The cytoskeleton was seen to be mainly composed of actin filaments forming dense networks. The network contained scattered star-like foci from which actin filaments radiated. When the ventral plasma membrane-cytoskeleton complex was further treated with Nonidet P-40, the membrane was dissolved to expose the glass surface with actin foci persisting on glass. When the complex was removed by further treatment with Nonidet P-40 and DNase I, the looped threads became visible. Confocal laser microscopy of glass-adherent macrophages stained with fluorescent phalloidin showed the preferential distribution of F-actin in the ventral cytoplasm along the plasma membrane, where intense fluorescent spots were also scattered. Confocal interference reflection microscopy revealed densely populated dark dots and striae of focal contact, which corresponded in overall distribution to actin foci and looped threads. These observations suggest that actin cytoskeleton is closely associated with looped threads to reinforce cell adhesion to glass.     

Differential Regulation of Focal Adhesion Kinase and Mitogen-Activated Protein Kinase Tyrosine Phosphorylation During Insulin-Like Growth Factor-I-Mediated Cytoskeletal Reorganization

Abstract: In SH-SY5Y human neuroblastoma cells, insulin-like growth factor (IGF)-I mediates membrane ruffling and growth cone extension. We have previously shown that IGF-I activates the tyrosine phosphorylation of focal adhesion kinase (FAK) and extracellular signal-regulated protein kinase (ERK) 2. In the current study, we examined which signaling pathway underlies IGF-I-mediated FAK phosphorylation and cytoskeletal changes and determined if an intact cytoskeleton was required for IGF-I signaling. Treatment of SH-SY5Y cells with cytochalasin D disrupted the actin cytoskeleton and prevented any morphological changes induced by IGF-I. Inhibitors of phosphatidylinositol 3-kinase (PI 3-K) blocked IGF-I-mediated changes in the actin cytoskeleton as measured by membrane ruffling. In contrast, PD98059, a selective inhibitor of ERK kinase, had no effect on IGF-I-induced membrane ruffling. In parallel with effects on the actin cytoskeleton, cytochalasin D and PI 3-K inhibitors blocked IGF-I-induced FAK tyrosine phosphorylation, whereas PD98059 had no effect. It is interesting that cytochalasin D did not block IGF-I-induced ERK2 tyrosine phosphorylation. Therefore, it is likely that FAK and ERK2 tyrosine phosphorylations are regulated by separate pathways during IGF-I signaling. Our study suggests that integrity as well as dynamic motility of the actin cytoskeleton mediated by PI 3-K is required for IGF-I-induced FAK tyrosine phosphorylation, but not for ERK2 activation.     

Calcium-dependant binding proteins associated with human placental syncytiotrophoblast microvillous cytoskeleton

Isolated human placental syncytiotrophoblast microvillous plasma membrane vesicles were extracted with Triton X-100 to yield a detergent-insoluble residue. The residue contained approx. 50% of the total membrane protein and was qualitatively different from untreated trophoblast on SDS-polyacrylamide gel electrophoresis, Western blots and dot-immunobinding assay. Three major proteins, with molecular weights of 68, 36 and 34 kDa, dissociated from this non-ionic detergent-insoluble submembranous cytoskeletal fraction in the presence of calcium chelators. They were immunologically related to human lymphocyte cytoskeletal calcium-binding proteins, and the 36 kDa component reacted with antisera to the phospholipase A2 inhibitor, lipocortin II. Anti-lipocortin I sera did not recognise the 34 kDa protein, but did react with a series of trophoblast cytoskeletal proteins in the 34-37 kDa region. Incubation of epidermal growth factor with isolated trophoblast membrane vesicles stimulated the phosphorylation of a 36 kDa protein on tyrosine residues. Immunoprecipitation studies further showed there was no phosphorylation of the 34 kDa protein, but the 68 kDa protein was a major phosphorylated component of isolated syncytiotrophoblast membranes. p68 was principally phosphorylated on serine with slight tyrosine phosphorylation which showed an apparent increase after epidermal growth factor treatment. These results indicate a family of calcium-dependant binding proteins, some of which are phosphorylated, associated with the submembranous cytoskeleton of syncytiotrophoblast microvilli.     

Vinculin is proteolyzed by calpain during platelet aggregation: 95 kDa cleavage fragment associates with the platelet cytoskeleton

The focal adhesion protein vinculin contributes to cell attachment and spreading through strengthening of mechanical interactions between cell cytoskeletal proteins and surface membrane glycoproteins. To investigate whether vinculin proteolysis plays a role in the influence vinculin exerts on the cytoskeleton, we studied the fate of vinculin in activated and aggregating platelets by Western blot analysis of the platelet lysate and the cytoskeletal fractions of differentially activated platelets. Vinculin was proteolyzed into at least three fragments (the major one being approximately 95 kDa) within 5 min of platelet activation with thrombin or calcium ionophore. The 95 kDa vinculin fragment shifted cellular compartments from the membrane skeletal fraction to the cortical cytoskeletal fraction of lysed platelets in a platelet aggregation-dependent manner. Vinculin cleavage was inhibited by calpeptin and E64d, indicating that the enzyme responsible for vinculin proteolysis is calpain. These calpain inhibitors also inhibited the translocation of full-length vinculin to the cytoskeleton. We conclude that cleavage of vinculin and association of vinculin cleavage fragment(s) with the platelet cytoskeleton is an activation response that may be important in the cytoskeletal remodeling of aggregating platelets.     

Supramolecular organization of the subsarcolemmal cytoskeleton of adult skeletal muscle fibers. A review

This review is focused on the composition and organization of the junctional subsarcolemmal cytoskeleton of adult muscle fibers. The cytoskeleton of muscle fibers is organized in functionally distinct compartments and the subsarcolemmal cytoskeleton itself can be broadly divided into junctional (myotendinous junction, neuromuscular junction and costameres) and non-junctional domains. In junctional zones three different multimolecular cytoskeletal complexes coexist: the focal adhesion-type, the spectrin-based and the dystrophin vs utrophin-based membrane skeleton systems. These complexes extend over several levels, from intracytoplasmic to subsarcolemmal and transmembranous; their common feature is the anchorage of actin filaments emanating from the intracytoplasmic level. The different cytoskeletal proteins, their putative roles and their interactions with various signaling pathways are presented here in detail. The subsarcolemmal cytoskeleton complexes are thought to play distinct physiological roles (membrane stabilization, force transmission to extracellular matrix, ionic channel anchorage, etc) but their colocalization on the three sarcolemmal junctional domains strongly suggests interrelated or common functions.     

Actin cytoskeletal modulation of pressure-induced depolarization and Ca(2+) influx in cerebral arteries

The objective of this study was to examine the role of the actin cytoskeleton in the development of pressure-induced membrane depolarization and Ca(2+) influx underlying myogenic constriction in cerebral arteries. Elevating intraluminal pressure from 10 to 60 mmHg induced membrane depolarization, increased intracellular cytosolic Ca(2+) concentration ([Ca(2+)](i)) and elicited myogenic constriction in both intact and denuded rat posterior cerebral arteries. Pretreatment with cytochalasin D (5 microM) or latrunculin A (3 microM) abolished constriction but enhanced the [Ca(2+)](i) response; similarly, acute application of cytochalasin D to vessels with tone, or in the presence of 60 mM K(+), elicited relaxation accompanied by an increase in [Ca(2+)](i). The effects of cytochalasin D were inhibited by nifedipine (3 microM), demonstrating that actin cytoskeletal disruption augments Ca(2+) influx through voltage-sensitive L-type Ca(2+) channels. Finally, pressure-induced depolarization was enhanced in the presence of cytochalasin D, further substantiating a role for the actin cytoskeleton in the modulation of ion channel function. Together, these results implicate vascular smooth muscle actin cytoskeletal dynamics in the control of cerebral artery diameter through their influence on membrane potential as well as via a direct effect on L-type Ca(2+) channels.     

Integrity of the cortical actin ring is required for activation of the PI3K/Akt and p38 MAPK signaling pathways in redifferentiation of chondrocytes on chitosan

Cell shape alterations and accompanying cytoskeletal changes have diverse effects on cell function. We have already shown that dedifferentiated chondrocytes have a round cell morphology and undergo redifferentiation when cultured on chitosan membrane. In the present study, we investigate the role of the cytoskeleton in chondrocyte redifferentiation. Chondrocytes obtained from a micromass culture of chick limb bud mesenchymal cells were subcultured four times. Immunofluorescence analysis of F-actin showed cortical distribution of the actin cytoskeleton upon subculture of dedifferentiated chondrocytes on chitosan membrane. Treatment with cytochalasin D disrupted the cortical actin ring formed during cultivation of chondrocytes on the chitosan membrane, and inhibited chondrocyte redifferentiation. Moreover, cytochalasin D inhibited the phosphorylation of Akt and p38 mitogen activated protein kinase (MAPK), induced during redifferentiation on chitosan membrane. LY294002, an inhibitor of phosphatidylinositol-3-OH-kinase (PI3K), suppressed chondrocyte redifferentiation. These findings suggest that integrity of the actin cytoskeleton is a crucial requirement for PI3K/Akt and p38 MAPK in chondrocyte redifferentiation.     

Association of vinculin to the platelet cytoskeleton during thrombin-induced aggregation

Vinculin is a protein generally believed to be involved in membrane-cytoskeleton interaction, and its presence in platelets has been verified earlier. Here we show that in resting bovine platelets, vinculin is not associated with the Triton-insoluble cytoskeletal fraction but becomes incorporated into it during the thrombin-induced activation process. The incorporation starts around the same time as the release reaction and only after the shape change and the first phase of aggregation have taken place. Its time course parallels the cytoskeletal association of actin and certain other contractile proteins. Vinculin is a minor component of platelet cytoskeleton and only about 10% of the total platelet vinculin becomes incorporated into the Triton X-100 residue.     

Translocation of pp60c-src to the cytoskeleton during platelet aggregation.

The high amount of pp60c-src in platelets has led to speculation that this kinase is responsible for tyrosine-specific phosphorylation of cellular proteins during platelet activation by different agonists, and is, therefore, implicated in signal transduction of these cells. Unlike pp60v-src, the association of which with the cytoskeleton appears to be a prerequisite for transformation, pp60c-src is detergent-soluble in fibroblasts overexpressing the c-src gene, and its role in normal cellular function remains elusive. To gain a better understanding of the function of pp60c-src we have investigated the subcellular distribution of pp60c-src and its relationship to the cytoskeleton during platelet activation. Quantitative immunoblotting and immunoprecipitation have revealed that pp60c-src is detergent-soluble in resting platelets, while 40% of total platelet pp60c-src becomes associated with the cytoskeletal fraction upon platelet activation. We have also shown that a small pool of pp60c-src is associated with the membrane skeletal fraction which remains unchanged during the activation process. The interaction of pp60c-src with cytoskeletal proteins strongly correlates with aggregation and is mediated by GPIIb/IIIa receptor-fibrinogen binding. We suggest that the translocation of pp60c-src to the cytoskeleton and its association with cytoskeletal proteins may regulate tyrosine phosphorylation in platelets.     

Many cytoskeletal proteins associate with the hela cytoskeleton during translation in vitro

Observations that cytoskeletal proteins assemble in vivo close to the time and site of synthesis have been confirmed and extended by an in vitro translation system. HeLa cytoskeletons prepared with Triton in a translation-extraction buffer without reticulocyte or wheat germ lysate efficiently incorporate 35S-methionine into polypeptides, and are stable during this translation. Cytoskeletal proteins translated in this way associate with the HeLa cytoskeleton independent of the concentration of soluble proteins. These associations are puromycin-resistant before the proteins are complete; the protein associations made in vitro show only minor differences from those made in vivo. The protein associations are not simply a consequence of protein solubility in the buffers used, as the associations require initiation in vivo. These results indicate that many cytoskeletal proteins associate with the cytoskeleton during translation.     

Roles of ATP and cytoskeleton in the regulation of Na+/H+ exchanger along the nephron luminal membrane

Although in LLC-PK cells ATP depletion has been shown to result in alterations of cytoskeleton actin and an inhibition of Na+/H+ exchanger activity, there is little information concerning the regulation of this exchanger in the distal luminal membrane by ATP and actin filaments. The present study examined the direct effect of ATP and cytochalasin B on the Na+/H+ exchanger activity in the proximal and distal tubule luminal membranes. The presence of 100 microM ATP in the luminal membrane vesicles from rabbit proximal tubules did not influence the Ethyl Isopropyl Amiloride sensitive Na+ uptake by these membranes. In contrast, the same treatment of luminal membranes from distal tubules significantly enhanced the exchanger activity from 0.22 +/- 0.04 to 0.39 +/- 0.08 pM/microg/10 sec (P < 0.02). When ATP was replaced by its nonhydrolysable form, ATPgammas, the effect on the distal luminal membrane was strongly diminished suggesting that the action of the nucleotide implicates a phosphorylation step. Confirming this hypothesis, addition of 300-microM-Rp cAMP, a protein kinase A inhibitor, completely abolished the effect of ATP. In view of the fact that a tight relationship has been described between ATP, the cytoskeleton complex and the exchanger activity, we studied the effect of cytochalasin B on this activity. The presence of 20 microM cytochalasin B in the distal luminal membrane vesicles induced, as observed with ATP, a significant increase in the Na+ uptake. However, the actions of ATP and cytochalasin B were not additive. These results suggest that firstly, ATP and short actin filaments of the cytoskeleton regulate the distal luminal isoform through an intramembranous mechanism and secondly, a phosphorylation mechanism is, at least partially, implicated in the action of ATP. In contrast, the proximal tubule exchanger is regulated through different mechanisms.     

Role of the calcium-calpain pathway in cytoskeletal damage after eccentric contractions.

The mechanism(s) underlying eccentric damage to skeletal muscle cytoskeleton remain unclear. We examined the role of Ca(2+) influx and subsequent calpain activation in eccentric damage to cytoskeletal proteins. Eccentric muscle damage was induced by stretching isolated mouse muscles by 20% of the optimal length in a series of 10 tetani. Muscle force and immunostaining of the cytoskeletal proteins desmin, dystrophin, and titin were measured at 5, 15, 30, and 60 min after eccentric contractions and compared with the control group that was subjected to 10 isometric contractions. A Ca(2+)-free solution and leupeptin (100 microM), a calpain inhibitor, were applied to explore the role of Ca(2+) and calpain, respectively, in eccentric muscle damage. After eccentric contractions, decreases in desmin and dystrophin immunostaining were apparent after 5 min that accelerated over the next 60 min. Increased titin immunostaining, thought to indicate damage to titin, was evident 10 min after stretch, and fibronectin entry, indicating membrane disruption, was evident 20 min after stretch. These markers of damage also increased in a time-dependent manner. Muscle force was reduced immediately after stretch and continued to fall, reaching 56 +/- 2% after 60 min. Reducing extracellular calcium to zero or applying leupeptin minimized the changes in immunostaining of cytoskeletal proteins, reduced membrane disruption, and improved the tetanic force. These results suggest that the cytoskeletal damage and membrane disruption were mediated primarily by increased Ca(2+) influx into muscle cells and subsequent activation of calpain.     

Effects of membrane lipids and -proteins and cytoskeletal proteins on the kinetics of cholesterol exchange between high density lipoprotein and human red blood cells, ghosts and microvesicles.

To better understand the effects of plasma membrane lipids and proteins and the cytoskeleton on the kinetics of cellular cholesterol efflux, the effects of (1), selectively depleting either sphingomyelin (SM) or phosphatidylcholine (PC); (2), cross-linking the cytoskeleton, and (3), removing certain cytoskeletal and integral membrane proteins on radiolabelled cholesterol efflux from red blood cells (RBC) have been studied. When RBC were treated with either phospholipase A2 or sphingomyelinase C to hydrolyze either 30-40% of the PC or 40-50% of the SM, respectively, the halftimes (t1/2) for cholesterol efflux to excess HDL3 were not significantly altered, with the values being 4.4 +/- 0.8 h or 3.7 +/- 0.4 h, respectively, compared to 4.6 +/- 0.6 h for control RBC. To investigate the effects of the cytoskeleton on the rate of free cholesterol (FC) desorption from the plasma membrane, the cytoskeletal proteins were cross-linked by either heat-treatment or exposure to diamide and cholesterol efflux from ghosts of these cells was measured. Cross-linking the cytoskeletal proteins by diamide treatment resulted in no significant change in t1/2 for treated (3.6 +/- 0.6 h) compared to control (4.2 +/- 0.4 h) ghosts: this suggests that the cytoskeleton does not play a large role in modulating cholesterol efflux. To investigate the effects of membrane proteins on cholesterol efflux, RBC microvesicles, containing mainly band 3 and 4 proteins and little of the cytoskeletal proteins, such as spectrin (bands 1,2) or actin (band 5), were obtained by incubation with the ionophore A23187. With excess HDL3 present, microvesicles exhibited a t1/2 of 4.2 +/- 1.9 h (compared to the t1/2 of 4.2 +/- 0.4 h for control ghosts). The results described in this paper suggest that neither changing the SM/PC ratio in the membrane nor cross-linking the cytoskeletal proteins nor removing the cytoskeleton changes the t1/2 for cholesterol efflux to excess HDL3. Presumably, the cholesterol-phospholipid interactions are insensitive to these perturbations in membrane structure.     

Association of intercellular adhesion molecule-1 (ICAM-1) with actin- containing cytoskeleton and alpha-actinin

We have studied the cytoskeletal association of intercellular adhesion molecule-1 (ICAM-1, CD54), an integral membrane protein that functions as a counterreceptor for leukocyte integrins (CD11/CD18). A linkage between ICAM-1 and cytoskeletal elements was suggested by studies showing a different ICAM-1 staining pattern for COS cells transfected with wild-type ICAM-1 or with an ICAM-1 construct that replaces the cytoplasmic and transmembrane domains of ICAM-1 with a glycophosphatidylinositol (GPI) anchor. Wild-type ICAM-1 appeared to localize most prominently in microvilli whereas GPI-ICAM-1 demonstrated a uniform cell surface distribution. Disruption of microfilaments with cytochalasin B (CCB) changed the localization of wild-type ICAM-1 but had no effect on GPI-ICAM-1. Some B-cell lines demonstrated a prominent accumulation of ICAM-1 into the uropod region whereas other cell surface proteins examined were not preferentially localized. CCB also induced redistribution of ICAM-1 in these cells. For characterization of cytoskeletal proteins interacting with ICAM-1, a 28-residue peptide that encompasses the entire predicted cytoplasmic domain (ICAM-1,478-505) was synthesized, coupled to Sepharose-4B, and used as an affinity matrix. One of the most predominant proteins eluted either with soluble ICAM-1,478-505-peptide or EDTA, was 100 kD, had a pI of 5.5, and in Western blots reacted with alpha-actinin antibodies. A direct association between alpha-actinin and ICAM-1 was demonstrated by binding of purified alpha-actinin to ICAM-1,478-505-peptide and to immunoaffinity purified ICAM-1 and by a strict colocalization of ICAM-1 with alpha-actinin, but not with the cytoskeletal proteins talin, tensin, and vinculin. The region of ICAM-1,478-505 interacting with alpha-actinin was mapped to the area close to the membrane spanning region. This region contains several positively charged residues and appears to mediate a charged interaction with alpha-actinin which is not highly dependent on the order of the residues.