Cytoskeletal Linker Protein Research Articles

Plectin 5'-transcript diversity: short alternative sequences determine stability of gene products, initiation of translation and subcellular localization of isoforms.

Plectin is a large cytoskeletal linker protein expressed as several different isoforms from a highly complex gene. This transcript diversity is mainly caused by short 5'-sequences contained in alternative first exons. To elucidate the influence of these sequence differences and to determine potential differential functionality of the resulting protein forms, we conducted a systematic investigation of plectin isoforms on transcript and protein levels. Isoform expression was highly dependent on the different 5' ends, largely due to effects of the 5'-untranslated regions. Initiation of translation downstream of the expected start site led to loss of actin- and integrin beta4-binding in some isoforms. The small alternative N-terminal sequences (5-180 residues) profoundly affected the subcelluar localization of this >500 kDa protein. Specifically, plectin 1f was concentrated at focal adhesion contacts and plectin 1b was exclusively targeted to mitochondria, providing a connection of these organelles to intermediate filaments. Thus, with plectin as a model, we demonstrate a role for 5'-untranslated regions and alternative 5'-splicing as an important regulatory mechanism of protein expression and protein function.

Production of the bullous pemphigoid antigen 230 (BP230) by Saccharomyces cerevisiae and Pichia pastoris

BP230 is a cytoskeletal linker protein of 2649 amino acids originally identified as the target autoantigen in bullous pemphigoid, a potentially devastating autoimmune skin blistering disorder. To better define its function, we sought to generate recombinant forms of BP230 in both Saccharomyces cerevisiae and Pichia pastoris after cloning its entire cDNA. By immunoblot analysis, full-length BP230 was not found in extracts of P. pastoris, whereas minor amounts of degraded BP230 were detected in extracts of S. cerevisiae. In contrast, both S. cerevisiae and P. pastoris were able to produce the 770-amino acid COOH-terminal domain of BP230. Furthermore, the production level of the recombinant BP230 tail in S. cerevisiae was significantly higher than that observed in P. pastoris and that of endogenous BP230 in cultured human keratinocytes. Finally, 12 of 17 (71%) BP sera recognized the recombinant BP230 protein in yeast extracts. Our results indicate that S. cerevisiae occasionally constitutes a better tool for recombinant protein production than P. pastoris. Although both its large size and poor solubility limit production of BP230, the developed yeast system provides cellular fractions enriched in BP230 recombinant proteins that constitute useful tools for the diagnosis of bullous pemphigoid.

A mosaic genetic screen for genes necessary for Drosophila mushroom body neuronal morphogenesis.

Neurons undergo extensive morphogenesis during development. To systematically identify genes important for different aspects of neuronal morphogenesis, we performed a genetic screen using the MARCM system in the mushroom body (MB) neurons of the Drosophila brain. Mutations on the right arm of chromosome 2 (which contains approximately 20% of the Drosophila genome) were made homozygous in a small subset of uniquely labeled MB neurons. Independently mutagenized chromosomes (4600) were screened, yielding defects in neuroblast proliferation, cell size, membrane trafficking, and axon and dendrite morphogenesis. We report mutations that affect these different aspects of morphogenesis and phenotypically characterize a subset. We found that roadblock, which encodes a dynein light chain, exhibits reduced cell number in neuroblast clones, reduced dendritic complexity and defective axonal transport. These phenotypes are nearly identical to mutations in dynein heavy chain Dhc64 and in Lis1, the Drosophila homolog of human lissencephaly 1, reinforcing the role of the dynein complex in cell proliferation, dendritic morphogenesis and axonal transport. Phenotypic analysis of short stop/kakapo, which encodes a large cytoskeletal linker protein, reveals a novel function in regulating microtubule polarity in neurons. MB neurons mutant for flamingo, which encodes a seven transmembrane cadherin, extend processes beyond their wild-type dendritic territories. Overexpression of Flamingo results in axon retraction. Our results suggest that most genes involved in neuronal morphogenesis play multiple roles in different aspects of neural development, rather than performing a dedicated function limited to a specific process.

Role of the Extracellular and Cytoplasmic Domains of CD44 in the Rolling Interaction of Lymphoid Cells with Hyaluronan under Physiologic Flow

CD44 can function as an adhesion receptor that mediates leukocyte rolling on hyaluronan (HA). To study the contributions of different domains of the standard isoform of CD44 to cell rolling, a CD44-negative mouse T lymphoma AKR1 was transfected with wild type (WT) or mutated cDNA constructs. A parallel flow chamber was used to study the rolling behavior of CD44 transfectants on immobilized HA. For CD44WT transfectants, the fraction of cells that rolled and the rolling velocity was inversely proportional to the amount of cell surface CD44. When the cytoplasmic domain distal to Gly(305) or sequences that serve as binding sites for cytoskeletal linker proteins, were deleted or replaced with foreign sequences, no significant changes in the rolling behavior of mutant cells, compared with the transfectant expressing CD44WT, were observed. Transfectants lacking 64 amino acids of the cytoplasmic tail distal to Cys(295) adhered to HA but showed enhanced rolling at low shear forces. When 83 amino acids from the "non-conserved" membrane-proximal region of the CD44 extracellular domain were deleted, cells adhered firmly to the HA substrate and did not roll at any fluid shear force tested. Unlike wild type cells that exhibited a nearly homogeneous distribution of CD44 on a smooth cell surface, cells expressing the non-conserved region deletion mutant accumulated CD44 in membrane protrusions. Disruption of the actin cytoskeleton with cytochalasin B precluded the formation of membrane protrusions, however, treated cells still adhered firmly to HA and did not roll. We conclude that interaction between the cytoplasmic domain of CD44 and the cytoskeleton is not required for cell rolling on immobilized ligand. The strong effect of deletion of the non-conserved region of the extracellular domain argues for a critical role of this region in CD44-dependent rolling and adhesion interactions with HA under flow.

PACE-1, a novel protein that interacts with the C-terminal domain of ezrin

The ERM proteins (ezrin, radixin, moesin) together with merlin comprise a subgroup of the band 4.1 superfamily. These proteins act as membrane cytoskeletal linker proteins mediating interactions between the cytoplasmic domains of transmembrane proteins and actin. To better understand how the ERM proteins function to regulate these junctional complexes, a yeast 2-hybrid screen was undertaken using ezrin as a bait. We describe here the identification and cloning of a novel protein, PACE-1, which binds to the C-terminal domain of ezrin. Characterization of PACE-1 in human breast cancer cell lines demonstrates it to have two distinct intracellular localizations. A proportion of the protein is associated with the cytoplasmic face of the Golgi apparatus. This distribution is dependent upon the presence of the PACE-1 N-terminal myristoylation consensus sequence but is not dependent on an association with ezrin. In contrast, PACE-1 colocalises with ezrin in the lamellipodia, where ezrin has a role in cell spreading and motility. A notable feature of PACE-1 is the presence of a putative N-terminal kinase domain; however, in biochemical assays PACE-1 was shown to have associated rather than intrinsic kinase activity. Together these data suggest that PACE-1 may play a role in regulating cell adhesion/migration complexes in migrating cells.

Protein evolution: structure‐function relationships of the oncogene beta‐catenin in the evolution of multicellular animals

Beta-catenin functions as a cytoskeletal linker protein in cadherin-mediated adhesion and as a signal mediator in wnt-signal transduction pathways. We use a novel integrative approach, combining evolutionary, genomic, and three-dimensional structural data to analyze and trace the structural and functional evolution of beta-catenin genes. This approach also enabled us to examine the effects of gene duplication on the structure and function of beta-catenin genes in Drosophila, C. elegans, and vertebrates. By sampling a large number of different taxa, we identified both ancestral and derived motifs and residues within the different regions of the beta-catenin proteins. Projecting amino acid substitutions onto the three- dimensional structure established for mouse beta-catenin, we identified specific domains that exhibit loss and gain of selective constraints during beta catenin evolution. Structural changes, changes in the amino acid substitution rate, and the appearance of novel functional domains in beta-catenin can be mapped to specific branches on the metazoan tree. Together, our analyses suggest that a single, beta-catenin gene fulfilled both adhesion and signaling functions in the last common ancestor of metazoans some 700 million years ago. In addition, gene duplications facilitated the evolution of beta-catenins with novel functions and allowed the evolution of multiple, single-function proteins (cell adhesion or wnt-signaling) from the ancestral, dual-function protein. Integrative methods such as those we have applied here, utilizing the 'natural experiments' present in animal diversity, can be employed to identify novel and shared functional motifs and residues in virtually any protein among the proteomes of model systems and humans.

Plectin tethers desmin intermediate filaments onto subsarcolemmal dense plaques containing dystrophin and vinculin

Plectin is a versatile cytoskeletal linker protein that preferentially localizes at interfaces between intermediate filaments and the plasma membrane in muscle, epithelial cells, and other tissues. Its deficiency causes muscular dystrophy with epidermolysis bullosa simplex. To better understand the functional roles of plectin beneath the sarcolemma of skeletal muscles and to gain some insights into the underlying mechanism of plectin-deficient muscular dystrophy, we studied in vivo structural and molecular relationships of plectin to subsarcolemmal cytoskeletal components, such as desmin, dystrophin, and vinculin, in rat skeletal muscles. Immunogold electron microscopy revealed that plectin fine threads tethered desmin intermediate filaments onto subsarcolemmal dense plaques overlying Z-lines and I-bands. These dense plaques were found to contain dystrophin and vinculin, and thus may be the structural basis of costameres. The in vivo association of plectin with desmin, (meta-)vinculin, dystrophin, and actin was demonstrated by immunoprecipitation experiments. Treatment of plectin immunoprecipitates with gelsolin reduced actin, dystrophin, and (meta-)vinculin but not desmin, implicating that subsarcolemmal actin could partly mediate the interaction between plectin and dystrophin or (meta-)vinculin. Altogether, our data suggest that plectin, along with desmin intermediate filaments, might serve a vital structural role in the stabilization of the subsarcolemmal cytoskeleton.

Tissue-specific regulation by estrogen of ezrin and ezrin/radixin/moesin-binding protein 50.

The morphology and function of rat GH3 pituitary cells are profoundly affected by estradiol-17beta (E2), presumably due to changes in the profile of gene expression. We recently reported that a major target of E2 in these cells is the ezrin gene, which encodes a cytoskeletal linker protein that forms a complex with ezrin/radixin/ moesin-binding protein 50 (EBP50) in some cell types. Other studies have shown that EBP50 levels are increased by E2 in human breast and uterine tissue. Thus, we examined whether ezrin and EBP50 expression is coordinately increased by E2 in GH3 cells in vitro and rat pituitary glands in vivo. Ezrin levels are repressed by the steroidal antiestrogen, ICI 182780, and this effect is abrogated by E2 and the ERalpha-specific agonist, PPT, in GH3 cells. In contrast, EBP50 levels remained constant during these treatments. Ezrin and EBP50 did not display extensive colocalization. Moreover, ezrin was not co-immunoprecipitated by an EBP50 antibody in parental GH3 cells or in GH3 cells stably overexpressing EBP50, but was co-immunoprecipitated with EBP50 in human breast MCF-7 cells. Disruption of the actin cytoskeleton of GH3 cells changed the distribution of ezrin within subcellular fractions, but had no effect on EBP50. Finally, in juvenile female rats, E2 injections increased ezrin expression in the pituitary and uterus, but increased EBP50 expression only in the uterus. These findings demonstrate tissue specificity in the formation of ezrin-EBP50 complexes and in the regulation of EBP50 expression in estrogen-responsive tissues.

Attachment of the PSGL-1 cytoplasmic domain to the actin cytoskeleton is essential for leukocyte rolling on P-selectin

P-selectin glycoprotein ligand-1 (PSGL-1) serves as the leukocyte ligand for P-selectin, and many of the structural features of its ectodomain required for interactions with P-selectin have been uncovered. In contrast, the function of the highly conserved PSGL-1 cytoplasmic domain has not been explored. Stable transfectants expressing similar levels of either wild-type PSGL-1 or truncated PSGL-1 in which only 4 cytoplasmic residues were retained (designated PSGL-1 Delta cyto), were analyzed. Transfectants expressing full-length PSGL-1 rolled well on P-selectin. In contrast, rolling was almost completely absent in cells transfected with PSGL-1 Delta cyto, even at low shear. Importantly, cells expressing truncated PSGL-1 were able to bind soluble P-selectin and to bind COS cells overexpressing P-selectin, demonstrating that the P-selectin binding site on the PSGL-1 Delta cyto transfectants was intact and was capable of recognizing P-selectin. Impaired rolling by PSGL-1 Delta cyto transfectants was not due to alterations in subcellular localization because both wild-type and truncated PSGL-1 had similar surface distributions on K562 transfectants. Treatment of cells expressing native PSGL-1 with actin cytoskeletal toxins inhibited adhesion in a dose-dependent way. PSGL-1 was associated with the actin cytoskeleton, and this interaction was greatly impaired in PSGL-1 Delta cyto- expressing cells. The PSGL-1 cytoplasmic domain interacted selectively with the ezrin/radixin/moesin (ERM) protein moesin, but not with other ERM proteins or several other cytoskeletal linker proteins. Pharmacologic disruption of interactions between moesin and F-actin in cells expressing PSGL-1 resulted in a dose-dependent inhibition of rolling on P-selectin. Thus, attachment of PSGL-1 to the leukocyte cortical cytoskeleton is essential for leukocyte rolling on P-selectin.

A novel PKC-regulated mechanism controls CD44 ezrin association and directional cell motility.

The dynamic assembly and disassembly of membrane cytoskeleton junctional complexes is critical in cell migration. Here we describe a novel phosphorylation mechanism that regulates the hyaluronan receptor CD44. In resting cells, CD44 is constitutively phosphorylated at a single serine residue, Ser325. After protein kinase C is activated, a switch in phosphorylation results in CD44 being phosphorylated solely at an alternative residue, Ser291. Using fluorescence resonance energy transfer (FRET) monitored by fluorescence lifetime imaging microscopy (FLIM) and chemotaxis assays we show that phosphorylation of Ser291 modulates the interaction between CD44 and the cytoskeletal linker protein ezrin in vivo, and that this phosphorylation is critical for CD44-dependent directional cell motility.

Alterations in myelination in the central nervous system of dystonia musculorum mice

Dystonia musculorum (dt) is an autosomal recessive sensory neuropathy in mice resulting from a mutation in the gene encoding the cytoskeletal linker protein Bpag1. In addition to neurodegeneration, dt mice display myelination abnormalities in the peripheral nervous system. In this report we investigated whether myelination abnormalities are also present in the central nervous system of dt(Tg4) mice. Transcripts for both neural isoforms of Bpag1 (a1 and a2) were detected in optic nerves and spinal cords of wild-type mice. Light microscopy of resin-embedded thin sections revealed a reduction in myelinated axons in both optic nerves and spinal cords in dt(Tg4) mice. As well, hypermyelinated axons were detected in these tissues. Ultrastructural analysis of optic nerves and spinal cords from dt(Tg4) mice revealed an increase in the number of amyelinated axons, the presence of hypo- and hypermyelinated axons, and redundant myelin that course away from axons. Changes in the level of myelin proteins accompanied the morphological alterations. Myelin-associated glycoprotein levels were reduced in optic nerves of dt(Tg4) mice, and myelin basic protein levels were altered in optic nerves, sciatic nerves, and spinal cords of affected mice. Short-term cultures of oligodendrocytes derived from dt(Tg4) mice did not show morphological alterations.

Trypanin is a cytoskeletal linker protein and is required for cell motility in African trypanosomes.

The cytoskeleton of eukaryotic cells is comprised of a complex network of distinct but interconnected filament systems that function in cell division, cell motility, and subcellular trafficking of proteins and organelles. A gap in our understanding of this dynamic network is the identification of proteins that connect subsets of cytoskeletal structures. We previously discovered a family of cytoskeleton-associated proteins that includes GAS11, a candidate human tumor suppressor upregulated in growth-arrested cells, and trypanin, a component of the flagellar cytoskeleton of African trypanosomes. Although these proteins are intimately associated with the cytoskeleton, their function has yet to be determined. Here we use double-stranded RNA interference to block trypanin expression in Trypanosoma brucei, and demonstrate that this protein is required for directional cell motility. Trypanin(−) mutants have an active flagellum, but are unable to coordinate flagellar beat. As a consequence, they spin and tumble uncontrollably, occasionally moving backward. Immunofluorescence experiments demonstrate that trypanin is located along the flagellum/flagellum attachment zone and electron microscopic analysis revealed that cytoskeletal connections between the flagellar apparatus and subpellicular cytoskeleton are destabilized in trypanin(−) mutants. These results indicate that trypanin functions as a cytoskeletal linker protein and offer insights into the mechanisms of flagellum-based cell motility.

MACF1 gene structure: a hybrid of plectin and dystrophin.

Mammalian MACF1 (Macrophin1; previously named ACF7) is a giant cytoskeletal linker protein with three known isoforms that arise by alternative splicing. We isolated a 19.1-kb cDNA encoding a fourth isoform (MACF1-4) with a unique N-terminus. Instead of an N-terminal actin-binding domain found in the other three isoforms, MACF1-4 has eight plectin repeats. The MACF1 gene is located on human Chr 1p32, contains at least 102 exons, spans over 270 kb, and gives rise to four major isoforms with different N-termini. The genomic organization of the actin-binding domain is highly conserved in mammalian genes for both plectin and BPAG1. All eight plectin repeats are encoded by one large exon; this feature is similar to the genomic structure of plectin. The intron positions within spectrin repeats in MACF1 are very similar to those in the dystrophin gene. This demonstrates that MACF1 has characteristic features of genes for two classes of cytoskeletal proteins, i.e., plectin and dystrophin.

Gene targeting of envoplakin, a cytoskeletal linker protein and precursor of the epidermal cornified envelope.

Envoplakin, a member of the plakin family of cytoskeletal linker proteins, is localized in desmosomes of stratified epithelial cells and is a component of the epidermal cornified envelope. Gene targeting in mouse embryonic stem cells was used to generate a null allele of envoplakin. No envoplakin transcripts from the targeted allele could be detected in the skin of newborn mice. Mice homozygous for the targeted allele were born in the normal Mendelian ratio and were fertile. They did not develop any discernible pathological phenotype up to the age of 1 year. The ultrastructural appearance of cornified envelopes from adult epidermis was indistinguishable between wild-type and knockout mice, and there was no evidence that the absence of envoplakin affected the subcellular distribution of periplakin or desmoplakin, two other plakins found in desmosomes. The proportion of immature cornified envelopes in the epidermis of newborn mice was greater in envoplakin-null animals than in heterozygous littermates or wild-type mice, and the envelopes had a larger surface area. This correlated with a slight delay in barrier acquisition during embryonic development. We conclude that although envoplakin is part of the scaffolding on which the cornified envelope is assembled, it is not essential for envelope formation or epidermal barrier function.

Loss of glomerular foot processes is associated with uncoupling of podocalyxin from the actin cytoskeleton.

Podocalyxin (PC), the major sialoprotein of glomerular epithelial cells (GECs), helps maintain the characteristic architecture of the foot processes and the patency of the filtration slits. PC associates with actin via ezrin, a member of the ERM family of cytoskeletal linker proteins. Here we show that PC is linked to ezrin and the actin cytoskeleton via Na(+)/H(+)-exchanger regulatory factor 2 (NHERF2), a scaffold protein containing two PDZ (PSD-95/Dlg/ZO-1) domains and an ERM-binding region. The cytoplasmic tail of PC contains a C-terminal PDZ-binding motif (DTHL) that binds to the second PDZ domain of NHERF2 in yeast two-hybrid and in vitro pull-down assays. By immunocytochemistry NHERF2 colocalizes with PC and ezrin along the apical domain of the GEC plasma membrane. NHERF2 and ezrin form a multimeric complex with PC, as they coimmunoprecipitate with PC. The PC/NHERF2/ezrin complex interacts with the actin cytoskeleton, and this interaction is disrupted in GECs from puromycin aminonucleoside-, protamine sulfate-, or sialidase-treated rats, which show a dramatic loss of foot processes, comparable to that seen in the nephrotic syndrome. Thus NHERF2 appears to function as a scaffold protein linking PC to ezrin and the actin cytoskeleton. PC/NHERF2/ezrin/actin interactions are disrupted in pathologic conditions associated with changes in GEC foot processes, indicating their importance for maintaining the unique organization of this epithelium.

Glial cell line-derived neurotrophic factor-responsive and neurotrophin-3-responsive neurons require the cytoskeletal linker protein dystonin for postnatal survival.

We have investigated the fate of different neurotrophin-responsive subpopulations of dorsal root ganglion neurons in dystonia musculorum (dt) mice. These mice have a null mutation in the cytoskeletal linker protein, dystonin. Dystonin is expressed by all sensory neurons and cross links actin filaments, intermediate filaments, and microtubules. The dt mice undergo massive sensory neurodegeneration postnatally and die at around 4 weeks of age. We assessed the surviving and degenerating neuronal populations by comparing the dorsal root ganglion (DRG) neurons and central and peripheral projections in dt mice and wildtype mice. Large, neurofilament-H-positive neurons, many of which are muscle afferents and are neurotrophin-3 (NT-3)-responsive, were severely decreased in number in dt DRGs. The loss of muscle afferents was correlated with a degeneration of muscle spindles in skeletal muscle. Nerve growth factor (NGF)-responsive populations, which were visualized using calcitonin gene-related peptide and p75, appeared qualitatively normal in the lumbar spinal cord, DRG, and hindlimb skin. In contrast, glial cell line-derived neurotrophic factor (GDNF)-responsive populations, which were visualized using the isolectin B-4 and thiamine monophosphatase, were severely diminished in the lumbar spinal cord, DRG, and hindlimb skin. Analysis of NT-3, NGF, and GDNF mRNA levels using semiquantitative reverse transcriptase-polymerase chain reaction revealed normal trophin synthesis in the peripheral targets of dt mice, arguing against decreased trophic synthesis as a possible cause of neuronal degeneration. Thus, the absence of dystonin results in the selective survival of NGF-responsive neurons and the postnatal degeneration of many NT-3- and GDNF-responsive neurons. Our results reveal that the loss of this ubiquitously expressed cytoskeletal linker has diverse effects on sensory subpopulations. Moreover, we show that dystonin is critical for the maintenance of certain DRG neurons, and its function may be related to neurotrophic support.

Ezrin function is required for ROCK-mediated fibroblast transformation by the Net and Dbl oncogenes.

The small G protein RhoA and its GDP/GTP exchange factors (GEFs) Net and Dbl can transform NIH 3T3 fibroblasts, dependent on the activity of the RhoA effector kinase ROCK. We investigated the role of the cytoskeletal linker protein ezrin in this process. RhoA effector loop mutants which can bind ROCK induce relocalization of ezrin to dorsal actin-containing cell surface protrusions, as do Net and Dbl. Both processes are inhibited by the ROCK inhibitor Y27632, which also inhibits association of ezrin with the cytoskeleton, and phosphorylation of T567, conserved between ezrin and its relatives radixin and moesin. ROCK can phosphorylate the ezrin C-terminus in vitro. The ezrin mutant T567A cannot be relocalized by activated RhoA, Net or Dbl or by ROCK itself, and also inhibits RhoA-mediated contractility and focal adhesion formation. Moreover, ezrin T567A, but not wild-type ezrin, restores contact inhibition to Net- and Dbl-transformed cells, and inhibits the activity of Net and Ras in focus formation assays. These results implicate ROCK-mediated ezrin C-terminal phosphorylation in transformation by RhoGEFs.

Acf7 (MACF) is an actin and microtubule linker protein whose expression predominates in neural, muscle, and lung development.

Several proteins belonging to the plakin family of cytoskeletal linker proteins have recently been identified, including dystonin/Bpag1 and plectin. These proteins are unique in their abilities to form bridges between different cytoskeletal elements through specialized modular domains. We have previously reported the cloning and partial characterization of Acf7, a novel member of the plakin family. More recently, the full-length cDNA for mouse Acf7 has been reported. Acf7 has a hybrid composition, with extended homology to dystonin/Bpag1 and plectin in the N-terminal half, and to dystrophin in the central and C-terminal half. Recent studies have demonstrated that Acf7 has functional actin and microtubule binding domains. Here, we describe the developmental expression profile for mouse Acf7. RNA in situ hybridization experiments revealed Acf7 transcripts in the dermomyotome and neural fold of day 8.5 mouse embryos. Later in development, Acf7 expression was predominant in neural and muscle tissues and was strongly up-regulated just before birth in type II alveolar cells of the lung. Altogether, our results suggest that Acf7 functions as a versatile cytoskeletal linker protein and plays an important role in neural, muscle, and lung development.

Plakoglobin suppresses epithelial proliferation and hair growth in vivo.

Plakoglobin regulates cell adhesion by providing a modulatable connection between both classical and desmosomal cadherins and their respective cytoskeletal linker proteins. Both plakoglobin and the related protein beta-catenin are posttranscriptionally upregulated in response to Wnt-1 in cultured cells. Upregulation of beta-catenin has been implicated in potentiating hyperproliferation and tumor formation. To investigate the role of plakoglobin in these functions we expressed a full-length (PG) and an NH(2)-terminally truncated form of plakoglobin (DeltaN80PG) in mouse epidermis and hair follicles, tissues which undergo continuous and easily observed postnatal renewal and remodeling. Expression of these constructs results in stunted hair growth, a phenotype that has also been observed in transgenic mice expressing Wnt3 and Dvl2 (Millar et al. 1999). Hair follicles from PG and DeltaN80PG mice show premature termination of the growth phase (anagen) of the hair cycle, an event that is regulated in part by FGF5 (Hebert et al. 1994). The proliferative rate of the epidermal cells was reduced and apoptotic changes, which are associated with entry into the regressive phase of the hair follicle cycle (catagen), occurred earlier than usual.

An epidermal plakin that integrates actin and microtubule networks at cellular junctions.

Plakins are cytoskeletal linker proteins initially thought to interact exclusively with intermediate filaments (IFs), but recently were found to associate additionally with actin and microtubule networks. Here, we report on ACF7, a mammalian orthologue of the Drosophila kakapo plakin genetically involved in epidermal-muscle adhesion and neuromuscular junctions. While ACF7/kakapo is divergent from other plakins in its IF-binding domain, it has at least one actin (K(d) = 0.35 microM) and one microtubule (K(d) approximately 6 microM) binding domain. Similar to its fly counterpart, ACF7 is expressed in the epidermis. In well spread epidermal keratinocytes, ACF7 discontinuously decorates the cytoskeleton at the cell periphery, including microtubules (MTs) and actin filaments (AFs) that are aligned in parallel converging at focal contacts. Upon calcium induction of intercellular adhesion, ACF7 and the cytoskeleton reorganize at cell-cell borders but with different kinetics from adherens junctions and desmosomes. Treatments with cytoskeletal depolymerizing drugs reveal that ACF7's cytoskeletal association is dependent upon the microtubule network, but ACF7 also appears to stabilize actin at sites where microtubules and microfilaments meet. We posit that ACF7 may function in microtubule dynamics to facilitate actin-microtubule interactions at the cell periphery and to couple the microtubule network to cellular junctions. These attributes provide a clear explanation for the kakapo mutant phenotype in flies.