细胞膜蛋白与细胞骨架蛋白相互作用研究进展

细胞膜蛋白与胞浆骨架蛋白的相互作用对于维持细胞正常形态 ,细胞粘附与信号传导有重要作用。含有 4 .1 JEF结构域的蛋白 4 .1超家族与含有PDZ结构域的MAGUK蛋白家族能结合多种膜蛋白胞内区与胞浆蛋白 ,在膜蛋白与胞浆蛋白之间建立联系 ,对于细胞、细胞 -细胞间连接的正常结构与功能的维持有着重要作用。     

Membrane-Associated Cytoskeletal Proteins in Squid Giant Axons

Abstract: Cytoskeletal proteins (e.g., tubulin, actin, and neurofilament proteins) in the squid giant axon are separable into KF-soluble and -insoluble forms. The KF-insoluble cytoskeletal components appear to constitute the major proteins in the subaxolemmal fibrous network on the inner surface of the axon. These cytoskeletal proteins and the subaxolemmal network are both highly soluble in KI solutions. Whereas giant axons tolerate prolonged perfusions in KF solutions with no loss of excitable properties, a relatively short perfusion with KI solution completely eliminates the excitability of the axon. The loss of this excitability correlates with the simultaneous dissolution of the subaxolemmal network of cytoskeletal proteins and the release of its proteins into the perfusate. These data support the hypothesis that cytoskeletal proteins associated with the inner surface of the axolemma are involved in the regulation of axonal excitability.     

Computational Prediction of Candidate Proteins for S-Nitrosylation in Arabidopsis thaliana

Nitric oxide (NO) is an important signaling molecule that regulates many physiological processes in plants. One of the most important regulatory mechanisms of NO is S-nitrosylation—the covalent attachment of NO to cysteine residues. Although the involvement of cysteine S-nitrosylation in the regulation of protein functions is well established, its substrate specificity remains unknown. Identification of candidates for S-nitrosylation and their target cysteine residues is fundamental for studying the molecular mechanisms and regulatory roles of S-nitrosylation in plants. Several experimental methods that are based on the biotin switch have been developed to identify target proteins for S-nitrosylation. However, these methods have their limits. Thus, computational methods are attracting considerable attention for the identification of modification sites in proteins. Using GPS-SNO version 1.0, a recently developed S-nitrosylation site-prediction program, a set of 16,610 candidate proteins for S-nitrosylation containing 31,900 S-nitrosylation sites was isolated from the entire Arabidopsis proteome using the medium threshold. In the compartments “chloroplast,” “CUL4-RING ubiquitin ligase complex,” and “membrane” more than 70% of the proteins were identified as candidates for S-nitrosylation. The high number of identified candidates in the proteome reflects the importance of redox signaling in these compartments. An analysis of the functional distribution of the predicted candidates showed that proteins involved in signaling processes exhibited the highest prediction rate. In a set of 46 proteins, where 53 putative S-nitrosylation sites were already experimentally determined, the GPS-SNO program predicted 60 S-nitrosylation sites, but only 11 overlap with the results of the experimental approach. In general, a computer-assisted method for the prediction of targets for S-nitrosylation is a very good tool; however, further development, such as including the three dimensional structure of proteins in such analyses, would improve the identification of S-nitrosylation sites.     

原生动物的细胞骨架蛋白及其功能组件

目前在原生动物中发现了许多新的细胞骨架蛋白,如中心元蛋白、副鞭毛杆蛋白等。深入研究发现,原生动物的细胞骨架在细胞的模式形成,细胞核的遗传中也具有重要作用。从功能组件角度着眼研究细胞骨架的功能,将有助于了解细胞骨架的进化机制。     

LRCH Proteins: A Novel Family of Cytoskeletal Regulators

Background

Comparative genomics has revealed an unexpected level of conservation for gene products across the evolution of animal species. However, the molecular function of only a few proteins has been investigated experimentally, and the role of many animal proteins still remains unknown. Here we report the characterization of a novel family of evolutionary conserved proteins, which display specific features of cytoskeletal scaffolding proteins, referred to as LRCHs.

Principal Findings

Taking advantage of the existence of a single LRCH gene in flies, dLRCH, we explored its function in cultured cells, and show that dLRCH act to stabilize the cell cortex during cell division. dLRCH depletion leads to ectopic cortical blebs and alters positioning of the mitotic spindle. We further examined the consequences of dLRCH deletion throughout development and adult life. Although dLRCH is not essential for cell division in vivo, flies lacking dLRCH display a reduced fertility and fitness, particularly when raised at extreme temperatures.

Conclusion/Significance

These results support the idea that some cytoskeletal regulators are important to buffer environmental variations and ensure the proper execution of basic cellular processes, such as the control of cell shape, under environmental variations.     

Process Formation in Astrocytes: Modulation of Cytoskeletal Proteins

Studies on primary astrocytes cultured in vitro have shown that process formation involves changes in cytoskeletal proteins and release of tension on the substratum. Actin filament reorganization has previously been found to be the major cytoskeletal change occurring during process formation. These changes are relatively rapid with breakdown of the actin web and release of contacts occur within 15 min. of cyclic AMP treatment. The former is regulated by myosin light chain (MLC) and actin depolymerizing factor (ADF), with MLC involved in the initial release of contractile tension and ADF in both initial and longer term actin breakdown. Our results show that the dephosphorylation of MLC is due to the phosphorylation and inactivation of myosin light chain kinase (MLCK) in response to cyclic AMP. To further study the mechanisms underlying the process formation in astrocytes we used endothelin-1 (ET-1), a vasopeptide which has been shown to inhibit process formation in astrocytes and sodium fluoride which is a general phosphatase inhibitor. We observe an increase in phosphorylation of MLC on inhibition of process formation. To study the role of adhesion in process formation we used suspension cultures of astrocytes. Our results with the astrocytes in suspension suggest that the process formation in astrocytes is adhesion dependent and the changes in ADF and MLC occur only when there is process formation.     

Synthesis and Turnover of Cytoskeletal Proteins in Cultured Astrocytes

Abstract: We previously reported that the cytoskeleton of rat astrocytes in primary culture contains vimentin, glial fibrillary acidic protein (GFAP), and actin. These proteins were found in a fraction insoluble in Triton X-100 and thought to be assembled in filamentous structures. We now used primary astrocyte cultures to study the kinetics of synthesis and turnover of these cytoskeletal proteins. The intermediate filament proteins were among the most actively synthesized by astrocytes. High levels of synthesis were detectable by the third day of culture in the early log phase of growth, and the pattern of labeling at day 3 was similar to that at 14 days when the cultures had reached confluency. In short-term incorporation experiments vimentin, GFAP, and actin in the Triton-insoluble fraction were labeled within 5 min after exposure of the cultures to radioactive leucine. We did not detect any saturation of labeling for up to 6 h of incubation. The turnover of filament proteins studied by following the decay of radioactivity from prelabeled vimentin, GFAP, and cytoskeletal actin displayed biphasic decay kinetics for all three proteins. In the initial phase a fast-decaying pool with a half-life of 12–18 h contributed about 40% of the total activity in each protein. A major portion, about 60%, of each protein, however, decayed much more slowly, exhibiting a half-life of about 8 days.     

The Emerging Role of Cytoskeletal Proteins as Reliable Biomarkers

Cytoskeletal proteins are essential building blocks of cells. More than 100 cytoskeletal and cytoskeleton‐associated proteins are known and for some, their function and regulation are understood in great detail. Apart from cell shape and support, they facilitate many processes such as intracellular signaling and transport, and cancer related processes such as proliferation, migration, and invasion. During the last decade, comparative proteomic studies have identified cytoskeletal proteins as in vitro markers for tumor progression and metastasis. Here, these results are summarized and a number of unrelated studies are highlighted, identifying the same cytoskeletal proteins as potential biomarkers. These findings might indicate that the abundance of these potential markers of tumor progression is associated with the biological outcome and are independent of the cancer origin. This correlates well with recently published results from the Cancer Genome Atlas, indicating that cancers show remarkable similarities in their analyzed molecular information, independent of their organ of origin. It is postulated that the quantification of cytoskeletal proteins in healthy tissues, tumors, in adjacent tissues, and in stroma, is a great source of molecular information, which might not only be used to classify tumors, but more importantly to predict patients’ outcome or even best treatment choices.     

Distribution of Cytoskeletal Proteins in Neomycin-Induced Protrusions of Human Fibroblasts

The organization of actin, tubulin, and vimentin was studied in protruding lamellae of human fibroblasts induced by the aminoglycoside antibiotic neomycin, an inhibitor of the phosphatidylinositol cycle. Neomycin stimulates the simultaneous protrusion of lamellae in all treated cells, and the lamellae remain extended for about 15–20 min, before gradually withdrawing. The pattern and distribution of actin, tubulin, and vimentin during neomycin stimulation were analyzed by fluorescence and electron microscopy. F-actin in the newly formed lamellae is localized in a marginal band at the leading edge. Tubulin is colocalized with F-actin in the marginal band, but the newly formed lamellae are initially devoid of microtubules. Over a period of 10 to 20 min after the addition of neomycin, microtubules grow into the lamellae from the adjacent cytoplasm, while the intensity of tubulin staining of the marginal band decreases. Distribution of vimentin remains unchanged in neomycin-treated cells and vimentin filaments do not enter the new protrusions. Treatment of cells with colchicine and Taxol do not inhibit neomycin-induced protrusion but protrusions are no longer localized at the ends of cell processes and occur all around the cell periphery. We conclude that actin filaments are the major component of the cytoskeleton involved in generating protrusions. Microtubules and, possibly, intermediate filaments control the pattern of protrusions by their interaction with actin filaments.     

The Changes of Cytoskeletal Proteins in Plasma of Acrylamide-Induced Rats

Acrylamide (ACR) is a known industrial neurotoxic chemical that can induce neurodegeneration. Cytoskeletal protein aggregation is a pathological hallmark of neurodegenerative disorders. This study was an initial exploration on cytoskeletal proteins in plasma as potential biomarkers of ACR neurotoxicity. Low and high ACR groups received 20 mg/kg and 40 mg/kg ACR by intraperitoneal injection in adult Wistar rats and control group received physiological saline. Rats were all killed after 8 weeks to evaluate the levels of neurofilament(NF)-L, NF-M, NF-H, β-actin, α-tubulin, β-tubulin, tau, MAP₂ proteins in plasma using both SDS-PAGE and western blotting. Compared with the control, the levels of NF-L, NF-M, NF-H, β-actin, tau, MAP₂ proteins decreased and the level of α-tubulin increased in high ACR group, the levels of α-tubulin, β-tubulin and MAP₂ increased in low ACR group. The results suggested that the changes of these proteins might be relevant to the neurotoxicity of ACR. Some of the cytoskeletal proteins in plasma might be used as marker of biological effect in ACR induced neuropathy.     

Modulation of Phosphorylation of Neuronal Cytoskeletal Proteins by Neuronal Depolarization

1. The neuronal cytoskeletal protein tau and the carboxy tails of cytoskeletal proteins neurofilament-M (NF-M) and neurofilament-H (NF-H) are phosphorylated on serine residues by the cyclin-dependent kinase cdk-5.2. In aggregating neuronal–glial cultures we show that veratridine-mediated cation influx causes dephosphorylation of tau, NF-M and NF-H. Dephosphorylation was blocked specifically by cyclosporine A but not by okadiac acid at concentrations up to 200 nM.3. These results suggest that veratridine-triggered cation influx causes activation of PP-2B (calcineurin) leading to dephosphorylation of these cytoskeletal proteins.     

Participation of Septin Cytoskeletal Proteins in the Nervous System Functioning

Septins, the cytoskeletal proteins discovered in the 1970s in budding yeast cells, are currently detected in most postmitotic cells of animals and humans. In the last decade, significant progress has been made in understanding the biochemical properties of septins and their biological functions. An increasing number of studies show that these proteins play an important role in the development and physiology of specific tissues and organs. The review surveys classification, major functions and localization of septins in the nervous system of mammals and humans. Models describing the mechanisms of the septin involvement in the neurotransmitter secretion from nerve endings and the role of septins in the pathogenesis of various neurodegenerative diseases are discussed.     

Synthesis of Cytoskeletal Proteins in Bulk-Isolated Neuronal Perikarya Takashi Nakayama

Abstract: Neuronal perikarya were isolated from young rat brain by sucrose density gradient centrifugation of the tissue, dissociated with a low concentration of trypsin. The isolated cells retained their endogenous proteins, and were capable of active protein synthesis. After incubation with L-[³⁵S]methionine, perikarya were homogenised and separated into soluble and particulate fractions by centrifugation at 70,000 g. Newly synthesised polypeptides in each fraction were resolved by SDS-gel and two-dimensional gel electrophoresis coupled with fluorography. Neuronal perikarya synthesised predominantly actin, and α¹-, α² and β-tubulin. In addition, polypeptides with molecular weights of 35,000, 68,000 and 85,000 were heavily labelled. On two-dimensional electrophoresis, microheterogeneities were seen in soluble actin as well as in soluble tubulins, indicating that heterogeneities reported for brain actin and tubulins are inherent in neuronal actin and tubulins, but not owing to the heterogeneity of cells in the brain tissue. Structural differences between soluble tubulins and those associated with the particulate fraction were indicated by two-dimensional gel electrophoresis and also by one-dimensional peptide maps. The 68,000 molecular weight polypeptide synthesised in neuronal perikarya in vitro yielded a peptide map virtually identical with that generated from the major component of the neurofilament triplet polypeptides that were synthesised in situ. The 160,000 and 200,000 components of the neurofilament triplet were also synthesised in perikarya in vitro , but to disproportionately weaker extents compared with the 68,000 component.     

Androgen Regulates Gene Expression of Cytoskeletal Proteins in Adult Rat Motoneurons

Expression of β-actin and β-tubulin mRNA was examined in androgen-sensitive motoneurons of the spinal nucleus of the bulbocavernosus (SNB) in adult male rats by in situ hybridization histochemistry using complementary DNAs encoding chick β-actin and mouse β-tubulin, respectively. Both hybridizable β-actin and βtubulin mRNAs were localized in the somata and proximal dendrites of SNB motoneurons. Removal of androgen by castration significantly reduced the expression levels of both β-actin and β-tubulin mRNAs in the SNB motoneurons, whereas the changes were prevented by testosterone treatment. In contrast, castration or testosterone treatment induced little or no change in the expression levels of these mRNAs in the much less androgen-sensitive motoneurons of the retrodorsolateral nucleus (RDLN). These results suggest that androgen regulates the expression of β-actin and β-tubulin genes in the SNB motoneurons and may provide evidence for the molecular mechanisms of hormonally induced neuronal plasticity in the SNB motoneurons.     

Altered Axonal Transport of Cytoskeletal Proteins in the Mutant Diabetic Mouse

Polypeptides in the motor axons of the sciatic nerve in 120-day-old normal and diabetic mice C57BL/Ks (db/db) were labeled by injection of [35S]methionine into the ventral horn of the spinal cord. At 8, 15, and 25 days after the injection, the distribution of radiolabeled polypeptides along the sciatic nerve was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Four major radiolabeled polypeptides, tentatively identified as actin, tubulin, and the two lightest subunits of the neurofilament triplet, were studied in both diabetic and control mice. In the diabetic animals, the two polypeptides identified as actin and tubulin showed a reduction of average velocity of migration along the sciatic nerve, resulting in a higher fraction of radioactivity in the proximal part of the sciatic nerve, whereas the front of radioactivity (advancing at maximal velocity) moved at a normal rate. In contrast, both the average and maximal velocities of the two neurofilament subunits were slower in the diabetic mice than in the control mice. These results indicate that the axonal transport of the cytoskeletal proteins is differentially affected in the course of diabetic neuropathy, and may suggest that the impairment concerns mainly the proteins carried by the slowest component of axonal transport.     

Slow Axonal Transport Impairment of Cytoskeletal Proteins in Streptozociti-Induced Diabetic Neuropathy

The impairment of slow axonal transport of cytoskeletal proteins was studied in the sciatic nerves of streptozocin-diabetic rats. [35S]Methionine was unilaterally injected into the fourth lumbar ganglion and spinal cord, to label the sensory and motor axons, respectively, and then the polymerized elements of the cytoskeleton and the corresponding soluble proteins were analyzed separately. In addition, the pellet/supernatant ratio for tubulin and actin was also assessed. Our results indicate that the velocity of slow component a (SCa) of axonal transport, particularly that of neurofilaments, was strongly reduced (by 60%) in sensory axons. At the same time, a decreased pellet/supernatant ratio of tubulin, possibly owing to a depolymerization of stable microtubules, was also observed. The transport of slow component b (SCb) of axonal transport was also impaired, but the extent of this impairment could not be precisely evaluated. In contrast, motor axons showed little or no impairment of both SCa and SCb at the time studied, a result suggesting a delayed development of the neuropathy in motor axons.     

Changes in Organization and Axonal Transport of Cytoskeletal Proteins During Regeneration

Changes in solubility and transport rate of cytoskeletal proteins during regeneration were studied in the motor fibers of the rat sciatic nerve. Nerves were injured by freezing at the midthigh level either 1-2 weeks before (experiment I) or 1 week after radioactive labeling of the spinal cord with L-[35S]methionine (experiment II). Labeled proteins in 6-mm consecutive segments of the nerve 2 weeks after labeling were analyzed following fractionation into soluble and insoluble populations with 1% Triton at 4 degrees C. When axonal transport of newly synthesized cytoskeleton was examined in the regenerating nerve in experiment I, a new faster component enriched in soluble tubulin and actin was observed that was not present in the control nerve. The rate of the slower main component containing most of the insoluble tubulin and actin together with neurofilament proteins was not affected. A smaller but significant peak of radioactivity enriched in soluble tubulin and actin was also detected ahead of the main peak when the response of the preexisting cytoskeleton was examined in experiment II. It is thus concluded that during regeneration changes in the organization take place in both the newly synthesized and the preexisting axonal cytoskeleton, resulting in a selective acceleration in rate of transport of soluble tubulin and actin.     

Differences in the Membrane Skeleton Proteins of RBC of AMoL Patients

We report here the different components of erythrocyte membrane skeleton proteins between acute monocytic leukemic (AMoL) patients and normal people. The bands in the 2 region of ghost membrane from AMoL patients exhibited significant differences on SDS gel electrophoresis. Band 2.2 was found to be missing and a “new” band with molecular weight (MW) 161 000 appeared. Also band 4.9 was missing, and the amounts of spectrin, actin, and band 4.8 of AMoL patients were decreased markedly. No such alterations can be seen in normal individuals, even in acute myeloid leukemic (AML) and chronic myeloid leukemic (CML) patients     

Quantitative Analysis of Axonal Transport of Cytoskeletal Proteins in Chicken Oculomotor Nerve

We studied the axonal transport characteristics of major cytoskeletal proteins: tubulin, the 69,000 molecular weight protein of chicken neurofilaments, and actin. After intracerebral injection of [35S]methionine, we monitored the specific radioactivity of these proteins as they passed through a very short nerve segment of the chicken oculomotor nerve. Specific radioactivities were assessed by quantitative sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. The transport patterns obtained for tubulin and the neurofilament protein were very similar, corresponding to transport rate ranges of 1-15 and 1-10 mm/day, respectively. A narrower velocity range of 3 to 4.3 mm/day was found for actin. Tubulin and the neurofilament protein appeared to be largely dispersed during the course of their transit along the nerve. The radioactivity associated with the proteins studied persisted in the nerve segment for a long time after the bulk of the labeled molecules had swept down. Finally, none of these proteins was observed to be transported with the fast axonal transport.