细胞骨架对凝血酶诱导人血小板分泌反应的调节

本文利用血小板表面外露的ＧＭＰ－１４０为血小板分泌反应的特异性标志,通过放射免疫分析法定量测定血小板表面ＧＭＰ－１４０分子数,研究了细胞骨架抑制剂对凝血酶导血小板分泌反应的影响。结果表明,凝血酶激活使血小板表面ＧＭＰ－１４０的外露明显增加,反应迅速,并在一定范围内呈剂量和时间依赖性;而ＡＤＰ刺激则几乎不引起ＧＭＰ－１４０外露的增加。凝血酶激活前加入不同的细胞骨架抑制剂处理可产生不同的效应：细胞松驰     

p38信号通路参与LPS诱导的Raw264.7细胞骨架重构

应用免疫荧光标记、基因转染等方法,观察脂多糖（LPS）刺激对单核细胞系Raw264.7细胞骨架的影响,探讨p38家族不同亚型对LPS诱导的细胞骨架蛋白微管蛋白与肌动蛋白变化的调控作用结果显示,未受LPS刺激的细胞富含微管蛋白,微管蛋白交联形成辐射状的交联丝网,丝网在细胞中分布均匀;LPS刺激后,微管蛋白募集在细胞膜、核膜周围;p38α、p38β、p38γ亚型的特异性抑制剂FHPI对LPS诱导的微管蛋白募集无影响,而p38无活性突变体p38δ(AF)的基因转染,可抑制LPS诱导的细胞骨架微管蛋白的募集;肌动蛋白在静息的细胞内主要存在于细胞膜周围,LPS作用后,肌动蛋白在细胞中形成广泛分布的辐射状应激纤维;p38上游激酶活性诱变体MKK6b基因转染可诱导Raw细胞形成类似的应激纤维,而p38γ(AF)的基因转染,可抑制LPS诱导的细胞应激纤维的形成.上述结果表明,p38δ可能参与了LPS诱导的微管蛋白的重构;而LPS诱导Raw细胞应激纤维的形成,可能是通过p38γ蛋白激酶而发挥作用.     

P38在LPS诱导大鼠雪旺氏(Schwann) 细胞TNF-α表达中的作用

应用免疫荧光标记、基因转染等方法,观察脂多糖（LPS）刺激对单核细胞系Raw264.7细胞骨架的影响,探讨p38家族不同亚型对LPS诱导的细胞骨架蛋白微管蛋白与肌动蛋白变化的调控作用结果显示,未受LPS刺激的细胞富含微管蛋白,微管蛋白交联形成辐射状的交联丝网,丝网在细胞中分布均匀;LPS刺激后,微管蛋白募集在细胞膜、核膜周围;p38α、p38β、p38γ亚型的特异性抑制剂FHPI对LPS诱导的微管蛋白募集无影响,而p38无活性突变体p38δ(AF)的基因转染,可抑制LPS诱导的细胞骨架微管蛋白的募集;肌动蛋白在静息的细胞内主要存在于细胞膜周围,LPS作用后,肌动蛋白在细胞中形成广泛分布的辐射状应激纤维;p38上游激酶活性诱变体MKK6b基因转染可诱导Raw细胞形成类似的应激纤维,而p38γ(AF)的基因转染,可抑制LPS诱导的细胞应激纤维的形成.上述结果表明,p38δ可能参与了LPS诱导的微管蛋白的重构;而LPS诱导Raw细胞应激纤维的形成,可能是通过p38γ蛋白激酶而发挥作用.     

MACF1与成骨细胞骨架之间的相互关系研究

采用激光共聚焦显微术研究微管微丝交联因子（MACF1）与成骨样细胞（MD63及MC3T3）微丝／微管骨架、黏着斑之间的相互关系．结果表明,MACF1不连续地分布于微管纤维上,与微丝骨架部分共定位于胞质中,在很多的成骨细胞中可见MACF1分布于骨架相关的粘着斑处：细胞松弛素B影响了MACF1在成骨细胞中的分布,并有使其向细胞核周围及核内转位的趋势．秋水仙素对MACF1的分布无明显的影响．转染了siRNA—MACFl的MG．63细胞微丝骨架纤维分布不连续、微管骨架纤维分布紊乱．这些结果提示MACF1不仅起交联微丝及微管细胞骨架的作用．而且还可稳定细胞骨架：成骨细胞MACF1的分布更依赖于微丝骨架的完整性．     

细胞骨架与血糖调节

细胞骨架由微丝、微管和中间丝构成,参与血糖调节这一复杂的生理过程,在胰岛素分泌、胰岛素功能和糖代谢相关酶类的细胞内分布等方面具有重要的作用。本文将从以上三个方面,对细胞骨架与血糖调节的关系加以综述。     

Sindbis病毒的蛋白质合成与细胞骨架的关系

我们以Sindbis病毒感染BHK-21细胞为模式,研究了病毒的感染与细胞骨架的关系。结果显示:在病毒感染早期,细胞的蛋白质合成迅速被抑制,细胞的多聚核糖体(polysome)和mRNA从骨架上脱落,而病毒的RNA结合到骨架上。我们的结果还进一步表明,病毒的RNA是通过其3′-尾端与骨架结合的。另一方面在对Sindbis病毒非结构蛋白在体内与体外合成与加工的比较中,我们发现病毒蛋白在体外翻译加工的速度远低于体内,并且出现很多未成熟蛋白(premature protein),这种区别可能在某种程度上反应细胞骨架在蛋白质合成与加工中的作用。此外,在用秋水仙素和细胞松驰素B破坏微管和微丝后,病毒非结构蛋白的合成与加工没有明显变化,而结构蛋白的合成则受到明显的抑制。这表明病毒的两类蛋白的合成所依赖的细胞骨架成分可能有所不同,在结构蛋白合成过程中,微丝和微管起了重要作用,在非结构蛋白合成过程中,中间丝很可能起了重要作用。     

Wangzaozin A诱导人宫颈癌HeLa细胞胞质骨架重排及细胞增殖抑制

该文采用锥虫蓝排染法、吉姆萨染色、流式细胞术及免疫荧光技术研究了wangzaozin A诱导HeLa细胞3种细胞骨架的重排特征以及细胞增殖抑制效应。结果显示,0.8~2.0μmol/L的wangzaozin A可显著抑制HeLa细胞增殖,导致细胞G1期阻滞,使细胞形态变化显著,不对称长条形细胞数量增多,与细胞骨架抑制剂诱导效果相似;wangzaozin A可显著改变细胞微管和角蛋白纤维排布方向,并诱导微管和角蛋白纤维聚合,导致细胞平均荧光强度显著增高,但减少细胞应力纤维数量,使平均荧光强度明显下降,显示了浓度和时间依赖性,该结果与紫杉醇对HeLa细胞的作用效果相似。Western blot显示,wangzaozin A并没有显著改变细胞内角蛋白、β肌动蛋白和α-微管蛋白含量,表明wangzaozin A诱导3种骨架纤维量的改变与其细胞内单体蛋白表达量无关。结果说明,wangzaozin A诱导的细胞骨架重排严重干扰细胞稳态,导致细胞及细胞核形态显著改变、细胞增殖抑制,但该化合物诱导细胞骨架重排的直接靶向位点尚需进一步研究。     

微丝的基本性质与细胞核肌动蛋白

微丝,作为细胞骨架的重要成员,普遍存在于所有的真核细胞中。构成微丝的肌动蛋白,与肌球蛋白一起作用,使细胞产生和传导机械力,并促进细胞运动。尽管人们很早就已经认识到体细胞核中存在单体肌动蛋白,但细胞核中聚合的微丝如何动态调控及行使何种功能,仍存在争议。该文概述了微丝细胞骨架的基本性质和成核过程,并讨论细胞核内肌动蛋白的功能。     

孤雌活化诱导恢复的小鼠减数分裂期间细胞骨架的动态与作用

减数分裂的顺利完成是胞质分裂和核分裂在时间和空间上的协调结果,细胞骨架系统在减数分裂的一系列事件中具有重要的调节作用.实验通过孤雌活化诱导小鼠MⅡ期卵减数分裂恢复,采用激光共聚焦显微术检测了减数分裂期间的微管、微丝和核的动态变化,并通过细胞骨架药物处理,以分析微管和微丝在减数分裂事件中的不同作用.结果显示：纺锤体微管为核的定位、分离和运动所必需;纺锤体从与质膜平行旋转至与质膜垂直是极体排放的前提;微丝是控制纺锤体旋转的关键因素;纺锤体旋转完成后微丝随即解聚,不参与极体的最后排出,形成原核后再重新组装.     

荧光标记法检测不同毒物对细胞骨架的影响

细胞骨架(Cytoskeleton)主要由微管(Microtubule,MT)、微丝(Microfilament,MF)以及中间丝(Intermediate filament,IF)这三种类型组成。它们在细胞的形态维持、物质运输、信号转导、能量转换及细胞的运动和分裂等多个过程中发挥着重要的作用。其中,由肌动蛋白组成的微丝是真核细胞中含量最丰富的一种蛋白复合体,以解聚时的球状肌动蛋白G-actin(Globular actin)或聚合时的纤丝状肌动蛋白F-actin(Filamen-tous actin)形式存在。正常细胞中肌动蛋白两种形态的转换处于动态平衡,共同行使细胞的变形运动、胞质分裂、基质附着和胞间连接等多…     

Role of calcium and calpain in complement-induced vesiculation of the platelet plasma membrane and in the exposure of the platelet factor Va receptor

The role of calcium and intracellular calpains in the expression of platelet prothrombinase activity was investigated. Incubation of gel-filtered platelets with complement proteins C5b-9 resulted in alpha-granule and dense granule secretion and exposure of membrane binding sites for coagulation factors Va and Xa. This was accompanied by the release of microparticles from the cell surface that incorporated plasma membrane glycoproteins GP Ib, IIb, and IIIa and the alpha-granule membrane protein GMP-140. Generation of these membrane microparticles was dependent on the presence of extracellular calcium and was accompanied by proteolytic degradation of the cytoskeletal proteins, actin binding protein (ABP), talin, and myosin heavy chain. Microparticle formation was also detected when unstirred platelets were activated by thrombin plus collagen, although proteolysis of ABP, talin, or myosin was not observed. Preincorporation of the calpain inhibitor leupeptin into the platelet cytosol completely blocked C5b-9-induced proteolysis of ABP, talin, and myosin. However, inhibition of this calpain-mediated proteolysis had no effect on platelet secretion, the generation of microparticles, the exposure of membrane sites for factors Va and Xa, or the expression of prothrombinase activity. Furthermore, the microparticles that formed in the presence of leupeptin contained intact ABP, talin, and myosin heavy chain. Prior depletion of ATP with metabolic inhibitors eliminated all platelet responses to thrombin plus collagen, but did not affect C5b-9-induced microparticle formation or exposure of binding sites for factor Va on the microparticles. These data indicate that the formation of microparticles and the expression of platelet prothrombinase activity in response to C5b-9 are dependent upon an influx of calcium into the platelet cytosol, but do not require metabolic energy or calpain-mediated proteolysis of cytoskeletal proteins.     

Rap1B and Rap2B translocation to the cytoskeleton by von Willebrand factor involves FcgammaII receptor-mediated protein tyrosine phosphorylation

Stimulation of human platelets with von Willebrand factor (vWF) induced the translocation of the small GTPases Rap1B and Rap2B to the cytoskeleton. This effect was specifically prevented by an anti-glycoprotein Ib monoclonal antibody or by the omission of stirring, but was not affected by the peptide RGDS, which antagonizes binding of adhesive proteins to platelet integrins. Association of Rap2B with the cytoskeleton was very rapid, while translocation of Rap1B occurred in a later phase of platelet activation and was totally inhibited by cytochalasin D. vWF also induced the rapid tyrosine phosphorylation of several proteins that was prevented by the tyrosine kinases inhibitor genistein and by cAMP-increasing agents. Under these conditions, also the association of Rap1B and Rap2B with the cytoskeleton was prevented. Translocation of Rap proteins to the cytoskeleton induced by vWF, but not by thrombin, was inhibited by a monoclonal antibody against the FcgammaII receptor. The same antibody inhibited vWF-induced tyrosine phosphorylation of selected substrates with molecular masses of about 75, 95, and 150 kDa. Three of these substrates were identified as the tyrosine kinase pp72(syk), the phospholipase Cgamma2, and the inositol 5-phosphatase SHIP. Our results indicate that translocation of Rap1B and Rap2B to the cytoskeleton is regulated by tyrosine kinases and suggest a novel role for the FcgammaII receptor in the mechanism of platelet activation by vWF.     

Characterization of human platelet GMP-140 as a heparin-binding protein

Human platelet GMP-140 has been identified as a heparin-binding protein. Purified platelet GMP-140 bound to Heparin-Sepharose CL-6B and was eluted by approximately 0.5 M sodium chloride. Radioiodinated GMP-140 bound specifically and saturably to heparin immobilized on Matrex-Pel 102 beads. Binding of radioiodinated GMP-140 to heparin-Matrex-Pel 102 beads was divalent cation-independent and was strongly inhibited by excess fluid phase GMP-140 and heparin and by other sulfated glycans such as fucoidin and dextran-sulfate. Binding was not inhibited by chondroitins 4- and 6-sulfate or mannose 6-phosphate.     

Stimulated secretion of endothelial von Willebrand factor is accompanied by rapid redistribution to the cell surface of the intracellular granule membrane protein GMP-140

We have examined the cell activation-dependent redistribution of the intracellular granule membrane protein GMP-140 of human endothelial cells. By dual-label immunofluorescence, the distribution of GMP-140 within cultured human umbilical vein endothelial cells was found to coincide with the distribution of von Willebrand factor (vWF), suggesting that GMP-140 is located in the membranes of vWF-containing storage granules. Stimulation of vWF secretion resulted in an increase in GMP-140 on the cell surface, as detected by increased binding of the monoclonal antibody S12 which recognizes the extracytoplasmic domain of GMP-140. For each agonist tested (histamine, thrombin, phorbol 12-myristate 13-acetate, and the calcium ionophore A23187) a dose-dependent redistribution of GMP-140 to the endothelial surface was observed which closely paralleled the dose-dependent secretion of vWF into the cell supernatant. When cells were maximally stimulated by histamine in the presence of antibody S12, a 4-fold increase in S12 uptake by the cells was observed. This increase occurred rapidly and reached a plateau by 10 min. In contrast, when histamine-stimulated cells were first fixed with paraformaldehyde or chilled to 4 degrees C before addition of antibody S12, only a transient increase in cell surface GMP-140 was detected. Under these conditions of arrested membrane turnover during antibody binding, cell surface GMP-140 was maximal 3 min after histamine stimulation and then declined to control levels by 20 min. These data suggest that stimulated secretion of vWF from endothelial cells entails fusion of vWF-containing storage granules with the plasma membrane. Once inserted into the plasma membrane, GMP-140 is subsequently removed from the endothelial surface, most likely by an endocytic mechanism.     

Stimulus-secretion coupling in platelets. Effects of drugs on secretion on adenosine 5'-triphosphate.

The mechanism of stimulus-secretion coupling in platelets was investigated by observing the effects of drugs on the kinetics on ATP secretion induced by either thrombin or the divalent cation ionophore A23187. The actual secretion is the same with either of these agents, since the rate constants and activation energies of secretion are the same and since drugs that affect the final, enzyme-independent steps of thrombin-induced secretion have the same effect on ionophore-induced secretion. Drugs that affect early steps of thrombin-induced secretion have no effect on ionophore-induced secretion. Drugs that act through cAMP (PGE1, theophylline, dibutyryl-cAMP) slow an early step in the mechanism of thrombin-induced secretion and completely block at higher levels, with the required concentration of inhibitor dependent on thrombin concentration. The inhibition of rate appears to be all-or-none, with no intermediate rates observed. By replacing thrombin with trypsin, which makes it possible to observe a complete change in rate-determining step from an enzyme-dependent to an enzyme-independent platelet step, it was found that these drugs slow the rate only when the enzyme-independent step is rate determining. These drugs have no effect on A23187-induced secretion. It was concluded that cAMP inhibits at a step after the enzyme step but before the final step by interfering with transmission of the stimulus-secretion coupling signal. Disruption of microfilament function by cytochalasin B (10 muM) accelerates the rate of secretion induced by either thrombin or ionophore. The microtubule agents colchicine, vinblastine, and vincristine had effects only at concentrations above those usually considered necessary for the specific inhibition of microtubule function. Drugs that inhibit prostaglandin synthesis (aspirin, indomethacin, eicosatetraynoic acid), drugs that block ATP production (antimycin A, deoxyglucose), or several other drugs previously reported to inhibit platelet function had no effect on secretion.     

Cytoskeletal changes in platelets induced by thrombin and phorbol myristate acetate (PMA).

Changes in shape, and aggregation that accompanies platelet activation, are dependent on the assembly and reorganization of the cytoskeleton. To assess the changes in cytoskeleton induced by thrombin and PMA, suspensions of aspirin-treated,32P-prelabeled, washed pig platelets in Hepes buffer containing ADP scavengers were activated with thrombin, and with PMA, an activator of protein kinase C. The cytoskeletal fraction was prepared by adding Triton extraction buffer. The Triton-insoluble (cytoskeletal) fraction isolated by centrifugation was analysed by SDS-PAGE and autoradiography. Incorporation of actin into the Triton-insoluble fraction was used to quantify the formation of F-actin. Thrombin-stimulated platelet cytoskeletal composition was different from PMA-stimulated cytoskeletal composition. Thrombin-stimulated platelets contained not only the three major proteins: actin (43 kDa), myosin (200 kDa) and an actin-binding protein (250 kDa), but three additional proteins of Mr56 kDa, 80 kDa and 85 kDa in the cytoskeleton, which were induced in by thrombin dose-response relationship. In contrast, PMA-stimulated platelets only induced actin assembly, and the 56 kDa, 80 kDa and 85 kDa proteins were not found in the cytoskeletal fraction. Exposure of platelets to thrombin or PMA induced phosphorylation of pleckstrin parallel to actin assembly. Staurosporine, an inhibitor of protein kinase C, inhibited actin assembly and platelet aggregation induced by thrombin or PMA, but did not inhibit the incorporation of 56 kDa, 80 kDa and 85 kDa into the cytoskeletal fraction induced by thrombin. These three extra proteins seem to be unrelated to the induction of protein kinase C. We conclude that actin polymerization and platelet aggregation were induced by a mechanism dependent on protein kinase C, and suggest that thrombin-activated platelets aggregation could involve additional cytoskeletal components (56 kDa, 80 kDa, 85 kDa) of the cytoskeleton, which made stronger actin polymerization and platelet aggregation more.     

Downregulation of GMP-140 (CD62 or PADGEM) expression on platelets by N,N-dimethyl and N,N,N-trimethyl derivatives of sphingosine.

GMP-140 (CD62 or PADGEM), a member of the selectin family, is a membrane glycoprotein in secretory granules of platelets and endothelial cells. When these cells are activated by agonists such as thrombin or AMP, GMP-140 is rapidly redistributed to the cell surface. The carbohydrate epitope defined by GMP-140 was identified as sialosyl-Le(x) (as for ELAM-1), which may play an essential role in adhesion of leukocytes or tumor cells on endothelial cells, through aggregation with platelets. Redistribution of GMP-140 from alpha-granules of platelets to the cell surface, induced by thrombin and PMA, was strongly inhibited by preincubation of platelets with N,N-dimethylsphingosine (DMS) or N,N,N-trimethylsphingosine (TMS) at 10-20 microM concentration for a brief period (5 min). Inhibition of GMP-140 redistribution to the cell surface by DMS or TMS was also detected by a cell adhesion assay using HL60 cells, which highly express sialosyl-Le(x); i.e., HL60 cells adhered on platelets activated by thrombin or PMA but not on platelets which were briefly preincubated with DMS or TMS followed by activation. The inhibitory effect of DMS or TMS on GMP-140 redistribution is not due to cytotoxicity, since the TMS-treated platelets were fully capable of aggregating in the presence of ristocetin. Sphingosine (SPN) and protein kinase C inhibitors such as H-7 and calphostin C showed weaker inhibitory activity than DMS and TMS. Our results indicate that both DMS and TMS could be useful reagents to inhibit cell surface expression of crucial selectins which promote adhesion of Le(x-) or sialosyl-Le(x)-expressing cells with platelets and endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biochemical and immunohistochemical characteristics of CD62 and CD63 monoclonal antibodies. Expression of GMP-140 and LIMP-CD63 (CD63 antigen) in human lymphoid tissues.

During platelet secretion granule membrane glycoproteins are translocated to the plasma membrane. We report here the biochemical and immunohistochemical characterization of a panel of platelet-secretion-specific, CD62 and CD63 monoclonal antibodies (MoAb), which we raised to thrombin-activated platelets. The CD62 MoAb identify the alpha-granule membrane protein GMP-140, also designated platelet activation-dependent granule external membrane protein (PADGEM). The number of epitopes on thrombin-activated platelets ranged from 15,000 to 20,000. The CD63 MoAb recognize a 30-60 kDalton integral membrane protein of lysosomes. Due to its distinct localization, we have designated the CD63 antigen lysosome integral membrane protein, CD63 (LIMP-CD63). The number of epitopes on thrombin-activated platelets ranged from 9000 to 11,000. Expression of GMP-140, a member of the Selectin family (also referred as the LEC-CAM family) of adhesion molecules, and LIMP-CD63 was examined on human spleen, thymus and lymph node by immunohistochemistry. Both GMP-140 and LIMP-CD63 showed a wide distribution in lymphoid tissues; vascular endothelial cells and tissue compartments that were readily accessible to blood-borne components were uniformly positive for GMP-140 and LIMP-CD63. Furthermore, LIMP-CD63 was expressed in polymorphonuclear granulocytes and macrophages.     

Structural and biosynthetic studies of the granule membrane protein, GMP-140, from human platelets and endothelial cells

GMP-140 is an integral membrane glycoprotein of apparent Mr = 140,000 located in secretory storage granules of platelets and vascular endothelial cells. When these cells are activated, GMP-140 redistributes from the membrane of the granules to the plasma membrane. To gain insight into the potential function of GMP-140, we examined aspects of its structure and biosynthesis. The amino acid composition of platelet GMP-140 revealed elevated numbers of cystinyl (6.1%), prolinyl (7.2%), and tryptophanyl (2.1%) residues. GMP-140 contained 28.8% carbohydrate by weight, distributed among N-acetylneuraminic acid, neutral sugar, and N-acetylglucosamine residues. Enzymatic removal of N-linked oligosaccarides reduced the protein's apparent Mr by more than 50,000. The biosynthesis of GMP-140 in HEL cells, which share biochemical features with megakaryocytes, was studied by pulse-chase labeling with [35S]cysteine followed by immunoprecipitation. HEL cells synthesized a heterogeneous GMP-140 precursor of 98-125 kDa which converted to a mature 140-kDa form within 40-60 min. Removal of high mannose oligosaccarides by endo-beta-N-acetylglucosaminidase H treatment reduced the apparent Mr of the precursor but not the mature protein. Tunicamycin-treated HEL cells synthesized three to four precursors of 80-92 kDa, suggesting the possibility of heterogeneity of GMP-140 at the protein level. Exposure of activated platelets to proteases followed by Western blotting indicated that most of the mass of GMP-140 was located on the extracytoplasmic side of the membrane. Our studies indicate that GMP-140 is a cysteine-rich, heavily glycosylated protein with a large extracytoplasmic domain. These features are compatible with a receptor function for the molecule when it is exposed on the surface of activated platelets and endothelial cells.     

Topographic distribution of a granule membrane protein (GMP-140) that is expressed on the platelet surface after activation: an immunogold-surface replica study

Monoclonal and polyclonal antibodies have been developed that recognize a 140 kD glycoprotein on the plasma membrane of activated, but not unstimulated, platelets. This glycoprotein is found in resting platelets as an alpha-granule membrane protein and has therefore been named GMP-140. After thrombin stimulation, alpha-granules fuse with the surface-connected canalicular system and GMP-140 is redistributed to the plasma membrane. In the present study, we immunolabeled unstimulated and activated human platelets and analyzed the distribution of GMP-140 over broad expanses of the plasma membrane using surface replication techniques. Fixed platelets were allowed to settle onto poly-L-lysine-coated coverslips and immunolabeled with polyclonal anti-GMP-140, followed by protein A gold. After critical-point drying, rotary-shadowed surface replicas were made. GMP-140 was not present on the surfaces of unstimulated platelets, but thrombin stimulation resulted in the massive expression of GMP-140 on the cell surface, with the immunogold label monodispersed. In contrast, we recently found that GPIIb-IIIa, the fibrinogen receptor, is monodispersed on unstimulated platelets and clustered on activated platelets. Although GMP-140's hemostatic function is unknown, its monodispersed surface pattern implies significant differences form GPIIb-IIIa with respect to ligand binding and/or cytoskeletal interaction.