Plectin: homologous to the N-terminal domain of Kakapo
(2) Localization to junctions and connection with microtubules

The integrin alpha6 beta4 is a major component of hemidesmosomes, in which it mediates firm adhesion to laminin 5. Previous studies have shown that the incorporation of alpha6 beta4 into hemidesmosomes requires a 303 amino acid stretch of the cytoplasmic domain of beta4, comprising part of the first fibronectin type III (FNIII) repeat, the second FNIII repeat and the segment that connects the second to the third FNIII repeat (connecting segment). Sequences within beta4 that are critical for its localization in hemidesmosomes have been further defined. These sequences also induce the redistribution of HD1/plectin into junctional complexes containing the integrin alpha6 beta4 in COS-7 cells, transfected with cDNAs encoding alpha6A and beta4. Truncation of the cytoplasmic domain of beta4 after amino acids 1,382 or 1,355 in the connecting segment, by which a potential tyrosine activation motif (TAM) is removed, does not prevent the localization of alpha6 beta4 in hemidesmosomes in the rat bladder carcinoma cell line 804G and neither does it eliminate the ability of alpha6 beta4 to change the subcellular distribution of HD1/plectin in COS-7 cells. In contrast, beta4 subunits in which the entire connecting segment had been deleted or which are truncated after amino acid 1,328, which removes almost the complete segment, has lost both of these functions. Furthermore, when beta4 subunits with either a deletion of the second FNIII repeat or a small deletion in this repeat are co-expressed with alpha6, the integrins are not localized in hemidesmosomes and do not induce the redistribution of HD1/plectin in COS-7 cells. Finally, the fourth FNIII repeat of beta4 can not replace the second in either of these activities. These findings establish that a region in beta4, which encompasses the second FNIII repeat and a stretch of 27 amino acids (1,329-1,355) of the connecting segment, is critical for the localization of alpha6beta4 in hemidesmosomes and that it regulates the distribution of HD1/plectin (Niessen, 1997).

Plectin, a versatile cytoskeletal linker protein, has an important role in maintaining the structural integrity of diverse cells and tissues. Plectin (-/-) mice die 2-3 days after birth exhibiting skin blistering caused by degeneration of keratinocytes. Ultrastructurally, hemidesmosomes and desmosomes appear unaffected. In plectin-deficient mice, however, hemidesmosomes are significantly reduced in number and apparently their mechanical stability is altered. The skin phenotype of these mice is similar to that of patients suffering from epidermolysis bullosa simplex (EBS)-MD, a hereditary skin blistering disease with muscular dystrophy, caused by defects in the plectin gene. In addition, plectin (-/-) mice reveal abnormalities reminiscent of minicore myopathies in skeletal muscle and disintegration of intercalated discs in heart. These results clearly demonstrate a general role of plectin in the reinforcement of mechanically stressed cells. Plectin (-/-) mice will provide a useful tool for the study of EBS-MD, and possibly other types of plectin-related myopathies involving skeletal and cardiac muscle, in an organism amenable to genetic manipulation (Andra, 1997).

The integrin heterodimer alpha 6 beta 4 is expressed in many epithelia and in Schwann cells. In stratified epithelia, alpha 6 beta 4 couple with BPAG1-e and BPAG2 to form hemidesmosomes, attaching externally to laminin and internally to the keratin cytoskeleton. To explore the function of this atypical integrin, and its relation to conventional actin-associated integrins, the removal of the beta 4 gene was targetted in mice. Tissues that express alpha 6 beta 4 are grossly affected. Stratified tissues are devoid of hemidesmosomes, display only a very fragile attachment to the basal lamina, and exhibit signs of degeneration and tissue disorganization. Simple epithelia which express alpha 6 beta 4 are also defective in adherence, even though they do not form hemidesmosomes. In the absence of beta 4, alpha 6 is dramatically downregulated, and other integrins do not appear to compensate for the loss of this heterodimer. These data have important implications for understanding integrin function in cell-substratum adhesion, cell survival and differentiation, and for understanding the role of alpha 6 beta 4 in junctional epidermolysis bullosa, an often lethal human disorder with pathology similar to these mice (Dowling, 1996).

BPAG1 is the major antigenic determinant of autoimmune sera of bullous pemphigoid (BP) patients. It is made by stratified squamous epithelia, where it localizes to the inner surface of specialized integrin-mediated adherens junctions (hemidesmosomes). To explore the function of BPAG1 and its relation to BP, the removal of the BPAG1 gene was targetted in mice. Hemidesmosomes are otherwise normal, but they lack the inner plate and have no cytoskeleton attached. Though not affecting cell growth or substratum adhesion, this compromises mechanical integrity and influences migration. Unexpectedly, the mice also develop severe dystonia and sensory nerve degeneration typical of dystonia musculorum (dt/dt) mice. In at least one other strain of dt/dt mice, BPAG1 gene is defective (Guo, 1995).

Hemidesmosomes (HDs) are stable anchoring structures that mediate the link between the intermediate filament cytoskeleton and the cell substratum. The contribution of various segments of the beta4 integrin cytoplasmic domain in the formation of HDs in transient transfection studies was investigated using immortalized keratinocytes derived from an epidermolysis bullosa patient deficient in beta4 expression. The expression of wild-type beta4 restores the ability of the beta4-deficient cells to form HDs and distinct domains in the NH2- and COOH-terminal regions of the beta4 cytoplasmic domain are required for the localization of HD1/plectin and the bullous pemphigoid antigens 180 (BP180) and 230 (BP230) in these HDs. The tyrosine activation motif located in the connecting segment (CS) of the beta4 cytoplasmic domain is dispensable for HD formation, although it may be involved in the efficient localization of BP180. Using the yeast two-hybrid system, a direct interaction was demonstrated between beta4 and BP180 which involves sequences within the COOH-terminal part of the CS and the third fibronectin type III (FNIII) repeat. Immunoprecipitation studies using COS-7 cells transfected with cDNAs for alpha6 and beta4 and a mutant BP180 which lacks the collagenous extracellular domain confirms the interaction of beta4 with BP180. Nevertheless, beta4 mutants which contained the BP180-binding region, but lack sequences required for the localization of HD1/plectin, fail to localize BP180 in HDs. Additional yeast two- hybrid assays indicate that the 85 COOH-terminal residues of beta4 can interact with the first NH2-terminal pair of FNIII repeats and the CS, suggesting that the cytoplasmic domain of beta4 is folded back upon itself. Unfolding of the cytoplasmic domain may be part of a mechanism by which the interaction of beta4 with other hemidesmosomal components, e.g., BP180, is regulated (Schaapveld, 1998).

By immunogold labeling, it has been demonstrated that "millipede-like" structures seen previously in mammalian cell cytoskeletons after removal of actin by treatment with gelsolin are composed of the cores of vimentin IFs with sidearms containing plectin. These plectin sidearms connect IFs to microtubules, the actin-based cytoskeleton and possibly membrane components. Plectin binding to microtubules is significantly increased in cells from transgenic mice lacking IFs and is reversed by microinjection of exogenous vimentin. These results suggest the existence of a pool of plectin which preferentially associates with IFs but may also be competed for by microtubules. The association of IFs with microtubules do not show a preference for Glu-tubulin. Nor does it depend upon the presence of MAP4 since plectin links are retained after specific immunodepletion of MAP4. The association of IFs with stress fibers survive actin depletion by gelsolin suggesting that myosin II minifilaments or components closely associated with them may play a role as plectin targets. These results provide direct structural evidence for the hypothesis that plectin cross-links elements of the cytoskeleton thus leading to integration of the cytoplasm (Svitkina, 1996).

ACF7 is a member of the spectraplakin family of cytoskeletal crosslinking proteins possessing actin and microtubule binding domains. ACF7 is an essential integrator of MT-actin dynamics. In endodermal cells, ACF7 binds along microtubules but concentrates at their distal ends and at cell borders when polarized. In ACF7's absence, microtubules still bind EB1 and CLIP170, but they no longer grow along polarized actin bundles, nor do they pause and tether to actin-rich cortical sites. The consequences are less stable, long microtubules with skewed cytoplasmic trajectories and altered dynamic instability. In response to wounding, ACF7 null cultures activate polarizing signals, but fail to maintain them and coordinate migration. Rescue of these defects requires ACF7's actin and microtubule binding domains. Thus, spectraplakins are important for controlling microtubule dynamics and reinforcing links between microtubules and polarized F-actin, so that cellular polarization and coordinated cell movements can be sustained (Kodama, 2003).

In motile fibroblasts, stable microtubules (MTs) are oriented toward the leading edge of cells. How these polarized MT arrays are established and maintained, and the cellular processes they control, have been the subject of many investigations. Several MT 'plus-end-tracking proteins,' or +TIPs, have been proposed to regulate selective MT stabilization, including the CLASPs, a complex of CLIP-170, IQGAP1, activated Cdc42 or Rac1, a complex of APC, EB1, and mDia1, and the actin-MT crosslinking factor ACF7. By using mouse embryonic fibroblasts (MEFs) in a wound-healing assay, this study shows that CLASP2 is required for the formation of a stable, polarized MT array but that CLIP-170 and an APC-EB1 interaction are not essential. Persistent motility is also hampered in CLASP2-deficient MEFs. ACF7 regulates cortical CLASP localization in HeLa cells, indicating it acts upstream of CLASP2. Fluorescence-based approaches show that GFP-CLASP2 is immobilized in a bimodal manner in regions near cell edges. These results suggest that the regional immobilization of CLASP2 allows MT stabilization and promotes directionally persistent motility in fibroblasts (Drabek, 2005).

ACF7 regulates cytoskeletal-focal adhesion dynamics and migration and has ATPase activity

Coordinated interactions between microtubule (MT) and actin cytoskeletons are involved in many polarized cellular processes. Spectraplakins are enormous (>500 kDa) proteins able to bind both MTs and actin filaments (F-actin) directly. To elucidate the physiological significance and functions of mammalian spectraplakin ACF7, it was conditionally targeted in skin epidermis. Intriguingly, ACF7 deficiency compromises the targeting of microtubules along F-actin to focal adhesions (FAs), stabilizes FA-actin networks, and impairs epidermal migration. Exploring underlying mechanisms, it was shown that ACF7's binding domains for F-actin, MTs, and MT plus-end proteins are not sufficient to rescue the defects in FA-cytoskeletal dynamics and migration functions of ACF7 null keratinocytes. An intrinsic actin-regulated ATPase domain was uncovered in ACF7, and the domain was demonstrated to be both functional and essential for these roles. These findings provide insight into the functions of this important cytoskeletal crosslinking protein in regulating dynamic interactions between MTs and F-actin to sustain directional cell movement (Wu, 2008).

ACF7 localizes to the Golgi complex

MACF1 (microtubule actin crosslinking factor), also called ACF7 (actin crosslinking family 7) is a cytoskeletal linker protein that can associate with both actin filaments and microtubules. A novel alternatively spliced isoform of MACF1 has been identified. This isoform was called MACF1b and the original isoform was renamed MACF1a. MACF1b is identical to MACF1a, except that it has a region containing plakin (or plectin) repeats in the middle of the molecule. MACF1b is ubiquitously expressed in adult tissues with especially high levels in the lung. The subcellular localization of MACF1b proteins was studied in mammalian cell lines. In two lung cell lines, MACF1b was chiefly localized to the Golgi complex. Upon treatments that disrupt the Golgi complex, MACF1b redistributed into the cytosol, but remained co-localized with the dispersed Golgi ministacks. MACF1b proteins can be detected in the enriched Golgi fraction by western blotting. The domain of MACF1b that targets it to the Golgi was found at the N-terminal part of the region that contains the plakin repeats. Reducing the level of MACF1 proteins by small-interfering RNA resulted in the dispersal of the Golgi complex (Lin, 2005).

Dystrophin: homologous to the central domain of Kakapo

The complete sequence of the human Duchenne muscular dystrophy (DMD) cDNA has been determined. The 3685 encoded amino acids of the protein product, dystrophin, can be separated into four domains. The 240 amino acid N-terminal domain has been shown to be conserved with the actin-binding domain of alpha-actinin. A large second domain is predicted to be rod-shaped and formed by the succession of 25 triple-helical segments similar to the repeat domains of spectrin (see Drosophila alpha Spectrin). The repeat segment is followed by a cysteine-rich segment that is similar in part to the entire COOH domain of the Dictyostelium alpha-actinin, while the 420 amino acid C-terminal domain of dystrophin does not show any similarity to previously reported proteins. The functional significance of some of the domains is addressed relative to the phenotypic characteristics of some Becker muscular dystrophy patients. Dystrophin shares many features with the cytoskeletal protein spectrin and alpha-actinin and is a large structural protein that is likely to adopt a rod shape about 150 nm in length (Koenig, 1998).

The actin binding domain of ACF7 binds directly to the tetratricopeptide repeat domains of rapsyn

Formation of the neuromuscular junction requires the release of agrin from the presynaptic terminal of motor neurons. Clustering of acetylcholine receptors (AChRs) on the postsynaptic sarcolemma is initiated by agrin-dependent activation of the muscle-specific kinase. While the postsynaptic scaffolding protein rapsyn is vital for high density AChR aggregation, little is known about the mechanism through which AChRs are immobilized on the postsynaptic membrane. Ultrastructural and immunohistochemical studies of rat skeletal muscle have suggested that AChRs are anchored to a membrane-associated cytoskeleton that contains spectrin-like proteins and is thus similar to that of the human erythrocyte, membrane skeleton that clusters nicotinic acetylcholine receptors in muscle. A protein of the spectrin superfamily, ACF7 (also known as MACF), is being studied as a postsynaptic cytoskeletal component of the neuromuscular junction. ACF7 has multiple cytoskeleton-binding domains, including an N-terminal actin-binding domain that is postulated to interact with rapsyn, the scaffolding protein that binds directly to AChRs. To test this hypothesis, fragments of these molecules were co-expressed in cultured fibroblasts and their co-distribution and interaction was assessed using confocal microscopy and co-immunoprecipitation. The actin-binding domain of ACF7 specifically interacts with the tetratricopeptide repeat domains of rapsyn. Furthermore, using surface plasmon resonance and blot overlay it is shown that the actin-binding domain of ACF7 binds directly to rapsyn. These results suggest that, in mammalian skeletal muscle, AChRs are immobilized in the membrane through rapsyn-mediated anchoring to an ACF7-containing network that in turn is linked to the actin cytoskeleton (Antolik, 2007)

Growth-arrest-specific 2 (gas2): homologous to the C-terminal domain of Kakapo

In order to elucidate a possible role of growth arrest-specific (gas) genes in the regulation of tissue proliferation, their expression in keratinocytes isolated from murine back skin was analyzed. On the mRNA level gas1, gas5, and gas6 are significantly expressed whereas there was a relatively low expression of gas2, gas3, and gas4. Using keratinocytes fractionated according to their density results in subpopulations of cells: differentiating suprabasal cells in fractions I and II; proliferative basal cells in fractions IIIa, III and IV. Gas2 protein to be expressed more strongly in the proliferative cells than in the differentiating cells. Stimulation of hyperproliferation by 12-O-tetradecanoylphorbol-13-acetate (TPA) results in a transient increase of gas2 protein content concomitantly with the time of maximal cell renewal. In this respect the murine keratinocyte cell line MSCP5 resembles freshly isolated keratinocytes. There is a higher expression of gas2 protein during exponential growth than during growth arrest, induced either by serum starvation or by TGFbeta treatment. Since, in contrast to the results reported for 3T3 cells, growth arrest within these cells was not accompanied by an elevation of gas2 protein, a cell-specific regulation of its expression is suggested (Manzow, 1996).

The regulation of Gas2 protein biosynthesis reflects the pattern of mRNA expression: its relative level is tightly associated with growth arrest. Gas2 seems to be regulated also at the posttranslational level via a phosphorylation mechanism. Gas2 is well conserved during the evolution with the same apparent molecular mass (36 kD) between mouse and human. Gas2 is a component of the microfilament system. It colocalizes with actin fiber, at the cell border and also along the stress fiber, in growth-arrested NIH 3T3 cells. The pattern of distribution, detected in arrested cells, can also be observed in growing cells when they are microinjected with the purified GST-Gas2 protein. In none of the analyzed oncogene-transformed NIH 3T3 cell lines is Gas2 expression induced under serum starvation (Brancolini, 1992).

Growth arrest-specific (Gas2) protein is a component of the microfilament system, that is highly expressed in growth arrested mouse and human fibroblasts and is hyperphosphorylated upon serum stimulation of quiescent cells. The kinetics of Gas2 phosphorylation, during Go-->G1 transition, as induced by addition of 20% FCS to serum starved NIH 3T3 cells, is temporally coupled to the reorganization of actin cytoskeleton. To better dissect the relationship between Gas2 phosphorylation and the modification of the microfilament architecture specific stimuli were used for both membrane ruffling (PDGF and PMA) and stress fiber formation (L-alpha-lysophosphatidic acid LPA) . All of them, similarly to 20% FCS, are able to downregulate Gas2 biosynthesis. PDGF and PMA induce Gas2 hyperphosphorylation that is temporally coupled with the appearance of membrane ruffling where Gas2 localizes. On the other hand LPA, a specific stimulus for stress fiber formation, fails to induce a detectable Gas2 hyperphosphorylation. Thus, Gas2 hyperphosphorylation is specifically correlated with the formation of membrane ruffling possibly implying a role of Gas2 in this process (Brancolini, 1994).

Gas2, a component of the microfilament system, belongs to the class of gas genes whose expression is induced at growth arrest. After serum or growth factor addition to quiescent NIH 3T3 cells, Gas2 is hyperphosphorylated and relocalized at the membrane ruffles. By overexpressing gas2wt and a series of deletion mutants of the C-terminal region, its role in the organization of the actin cytoskeleton has been analyzed in different cell lines. Overexpression of Gas2 deleted at its C-terminal region (delta 276-314 and delta 236-314), but not its wild-type form, induces dramatic changes in the actin cytoskeleton and cell morphology. These effects are not due to interference of the deleted forms with the endogenous Gas2wt function but can be ascribed to a gain of function. During apoptosis the C-terminal domain of Gas2 is removed by proteolytic cleavage, resulting in a protein that is similar in size to the described delta 276-314. Moreover, by using in vitro mutagenesis, it has been demonstrated that the proteolytic processing of Gas2 during apoptosis is dependent on an aspartic acid residue at position 279. The evidence accumulated in this work could thus represent a first example of a mechanism linking apoptosis with the co-ordinated microfilament-dependent cell shape changes, as possibly mediated by an interleukin-1 beta-converting enzyme (ICE)-like dependent proteolytic cleavage of the Gas2 protein (Brancolini, 1995).

The growth-arrest-specific 2 (gas2) gene was initially identified on account of its high level of expression in murine fibroblasts under growth arrest conditions, followed by downregulation upon reentry into the cell cycle. In this study, the expression patterns of the gas2 gene and the Gas2 peptide were established in the developing limbs of 11.5- to 14. 5-day mouse embryos. gas2 is expressed in the interdigital tissues, the chondrogenic regions, and the myogenic regions. Low-density limb culture and Brdu incorporation assays reveal that gas2 might play an important role in regulating chondrocyte proliferation and differentiation. Moreover, it might play a similar role during limb myogenesis. In addition to chondrogenesis and myogeneis, gas2 is involved in the execution of the apoptotic program in hindlimb interdigital tissues-by acting as a death substrate for caspase enzymes. The interdigital tissues undergo apoptosis between 13.5 and 15.5 days. Exactly at these time points, the C-terminal domain of the Gas2 peptide is cleaved as revealed by Western blot analysis. Moreover, pro-caspase-3 (an enzyme that can process Gas2) is cleaved into its active form in the interdigital tissues. The addition of zVAD-fmk, a caspase enzyme inhibitor, to 12.5-day-old hindlimbs maintained in organ culture reveals that the treatment inhibits interdigital cell death. This inhibition correlates with the absence of the Gas2 peptide and pro-caspase-3 cleavage. The data suggest that Gas2 might be involved in the execution of the apoptotic process (Lee, 1999).

The human Gas2-related gene on chromosome 22 (hGAR22) encodes two alternatively spliced mRNA species. The longer mRNA encodes a protein with a deduced molecular mass of 36.3 kDa (GAR22alpha), whereas the shorter mRNA encodes a larger protein with a deduced molecular mass of 72.6 kDa (GAR22beta). Both hGAR22 proteins contain a calponin homology actin-binding domain and a Gas2-related microtubule-binding domain. Using rapid amplification of cDNA ends, the mouse orthologue of hGAR22, mGAR22, has been cloned; its protein products are extremely well conserved. A human Gas2-related gene on chromosome 17 (hGAR17) has been cloned. hGAR17 also encodes two protein isoforms. The overall cytoskeletal binding properties of the hGAR17 and hGAR22 proteins are remarkably similar. hGAR17 mRNA expression is limited to skeletal muscle. Although hGAR22 and mGAR22 mRNAs are expressed nearly ubiquitously, mGAR22 protein can only be detected in testis and brain. Furthermore, only the beta isoform is present in these tissues. GAR22beta expression is induced in a variety of cultured cells by growth arrest. The absolute amounts of GAR22beta protein expressed are low. The beta isoforms of hGAR17 and hGAR22 appear to be able to crosslink microtubules and microfilaments in transfected cells. This finding suggests that the physiological functions of these proteins may involve integration of these two components of the cytoskeleton (Goriounov, 2003).

Apoptosis is characterized by proteolysis of specific cellular proteins by a family of cystein proteases known as caspases. Gas2, a component of the microfilament system, is cleaved during apoptosis and the cleaved form specifically regulates microfilaments and cell shape changes. Gas2 is a substrate of caspase-3 but not of caspase-6. Proteolytic processing both in vitro and in vivo is dependent on aspartic residue 279. Gas2 cleavage is only partially impaired in apoptotic MCF-7 cells which lack caspase-3, thus indicating that different caspases can process Gas2 in vivo. In vitro Gas2 is processed, albeit with low affinity, by caspase-7 thus suggesting that this caspase could be responsible for the incomplete Gas2 processing observed in UV treated MCF-7 cells. In vivo proteolysis of Gas2 is detected at an early stage of the apoptotic process when the cells are still adherent on the substrate and it is coupled to the specific rearrangement of the microfilament characterizing cell death. Gas2 in vitro binds to F-actin, but this interaction is unaffected by the caspase-3 dependent proteolytic processing (Sgorbissa, 1999).

kakapo Evolutionary homologs part 1/2

kakapo: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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