Low Mr tropomyosin isoforms from chicken brain and intestinal epithelium have distinct actin-binding properties

Tropomyosin isoforms of the low Mr class were isolated from chicken intestinal epithelium and brain, and their physical and functional properties were characterized. Tropomyosin from each tissue contains four distinct polypeptides, all of about 32,000 daltons. In two-dimensional gels, brain tropomyosin contains two major and two minor polypeptides; the major epithelium isoforms coelectrophorese with the two minor brain isoforms. Conversely, only small amounts of the major brain isoforms are detected in the epithelium. Actin-binding properties of brain tropomyosin isoforms are distinct from those of the intestinal epithelium. At 2.5 mM MgCl2 and physiological ionic strength, the intestinal epithelial tropomyosin binds to filamentous actin with an apparent Ka of 8 X 10(6) M-1 whereas brain tropomyosin has an apparent Ka of 8 X 10(5) M-1. Tropomyosin from either tissue binds actin cooperatively with a Hill coefficient of 2.3 for intestinal epithelial cell and 1.95 for brain tropomyosin. Isoforms from both tissues exhibit reduced head-to-tail polymerizability as compared to muscle tropomyosin. The actin-binding properties of intestinal epithelial cell tropomyosin are therefore similar to those of the muscle tropomyosins even though the isoforms have lower molecular weight, a paracrystal structure, and reduced head-to-tail polymerizability typical of the other nonmuscle tropomyosins. These results indicate that a heterogeneity of functional properties may be expressed among the low Mr tropomyosin isoforms.     

Tropomyosin isoforms define distinct microfilament populations with different drug susceptibility

Two tropomyosin isoforms, human Tm5(NM1) and Tm3, were over-expressed in B35 rat neuro-epithelial cells to examine preferential associations between specific actin and tropomyosin isoforms and to determine the role tropomyosin isoforms play in regulating the drug susceptibility of actin filament populations. Immunofluorescence staining and Western blot analysis were used to study the organisation of specific filament populations and their response to treatment with two widely used actin-destabilising drugs, latrunculin A and cytochalasin D. In Tm5(NM1) cells, we observed large stress fibres which showed predominant co-localisation of beta-actin and low-molecular-weight gamma-tropomyosin isoforms. Tm3 cells had an abundance of cellular protrusions which contained both the beta- and gamma-actin isoforms, predominately populated by high-molecular-weight alpha- and beta-tropomyosin isoforms. The stress fibres observed in Tm5(NM1) cells were more resistant to both latrunculin A and cytochalasin D than filaments containing the high-molecular-weight tropomyosins observed in Tm3 cells. Knockdown of the over-expressed Tm5(NM1) isoform with a human-specific Tm5(NM1) siRNA reversed the phenotype and caused a reversal in the observed drug resistance. We conclude that there are preferential associations between specific actin and tropomyosin isoforms, which are cell type specific, but it is the tropomyosin composition of a filament population which determines the susceptibility to actin-targeting drugs.     

A major transmembrane protein of Golgi-derived COPI-coated vesicles involved in coatomer binding

Formation of non-clathrin-coated vesicles requires the recruitment of several cytosolic factors to the Golgi membrane. To identify membrane proteins involved in this budding process, a highly abundant type I transmembrane protein (p23) was isolated from mammalian Golgi-derived COPI-coated vesicles, and its cDNA was cloned and sequenced. It belongs to the p24 family of proteins involved in the budding of transport vesicles (Stamnes, M.A., M.W. Craighead, M.H. Hoe, N. Lampen, S. Geromanos, P. Tempst, and J.E. Rothman. 1995. Proc. Natl. Acad. Sci. USA. 92:8011-8015). p23 consists of a large NH2-terminal luminal domain and a short COOH-terminal cytoplasmic tail (-LRRFFKAKKLIE-CO2-) that shows similarity, but not identity, with the sequence motif-KKXX-CO2-, known as a signal for retrieval of escaped ER-resident membrane proteins (Jackson, M.R., T. Nilsson, and P.A. Peterson. 1990. EMBO (Eur. Mol. Biol. Organ.) J. 9:3153-3162; Nilsson, T., M. Jackson, and P.A. Peterson. 1989. Cell. 58:707-718). The cytoplasmic tail of p23 binds to coatomer with similar efficiency as known KKXX motifs. However, the p23 tail differs from the KKXX motif in having an additional motif needed for binding of coatomer. p23 is localized to Golgi cisternae and, during vesicle formation, it concentrates into COPI-coated buds and vesicles. Biochemical analysis revealed that p23 is enriched in vesicles by a factor of approximately 20, as compared with the donor Golgi fraction, and is present in amounts stoichiometric to the small GTP-binding protein ADP-ribosylation factor (ARF) and coatomer. From these data we conclude that p23 represents a Golgi- specific receptor for coatomer involved in the formation of COPI-coated vesicles.     

Auxilin is required for formation of Golgi-derived clathrin-coated vesicles during Drosophila spermatogenesis

Clathrin has previously been implicated in Drosophila male fertility and spermatid individualization. To understand further the role of membrane transport in this process, we analyzed the phenotypes of mutations in Drosophila auxilin (aux), a regulator of clathrin function, in spermatogenesis. Like partial loss-of-function Clathrin heavy chain (Chc) mutants, aux mutant males are sterile and produce no mature sperm. The reproductive defects of aux males were rescued by male germ cell-specific expression of aux, indicating that auxilin function is required autonomously in the germ cells. Furthermore, this rescue depends on both the clathrin-binding and J domains, suggesting that the ability of Aux to bind clathrin and the Hsc70 ATPase is essential for sperm formation. aux mutant spermatids show a deficit in formation of the plasma membrane during elongation, which probably disrupts the subsequent coordinated migration of investment cones during individualization. In wild-type germ cells, GFP-tagged clathrin localized to clusters of vesicular structures near the Golgi. These structures also contained the Golgi-associated clathrin adaptor AP-1, suggesting that they were Golgi-derived. By contrast, in aux mutant cells, clathrin localized to abnormal patches surrounding the Golgi and its colocalization with AP-1 was disrupted. Based on these results, we propose that Golgi-derived clathrin-positive vesicles are normally required for sustaining the plasma membrane increase necessary for spermatid differentiation. Our data suggest that Aux participates in forming these Golgi-derived clathrin-positive vesicles and that Aux, therefore, has a role in the secretory pathway.     

Polarized human cholangiocytes release distinct populations of apical and basolateral small extracellular vesicles

Intercellular communication is critical for organismal homeostasis, and defects can contribute to human disease states. Polarized epithelial cells execute distinct signaling agendas via apical and basolateral surfaces to communicate with different cell types. Small extracellular vesicles (sEVs), including exosomes and small microvesicles, represent an understudied form of intercellular communication in polarized cells. Human cholangiocytes, epithelial cells lining bile ducts, were cultured as polarized epithelia in a Transwell system as a model with which to study polarized sEV communication. Characterization of isolated apically and basolaterally released EVs revealed enrichment in sEVs. However, differences in apical and basolateral sEV composition and numbers were observed. Genetic or pharmacological perturbation of cellular machinery involved in the biogenesis of intralumenal vesicles at endosomes (the source of exosomes) revealed general and domain-specific effects on sEV biogenesis/release. Additionally, analyses of signaling revealed distinct profiles of activation depending on sEV population, target cell, and the function of the endosomal sorting complex required for transport (ESCRT)-associated factor ALG-2–interacting protein X (ALIX) within the donor cells. These results support the conclusion that polarized cholangiocytes release distinct sEV pools to mediate communication via their apical and basolateral domains and suggest that defective ESCRT function may contribute to disease states through altered sEV signaling.     

Structural relationships of actin-binding proteins.

The sequences of a large number of actin-binding proteins have been compared. These findings, together with the results of protein-chemical analysis, peptide synthesis and site-directed and deletion mutagenesis, have led to the assignment of actin-binding sites. Within these segments, small actin-binding motifs have been delineated. Most actin-binding proteins interact with actin subdomain-1 but our analyses reveal neither primary nor secondary structure homology among these proteins, suggesting that actin binding does not follow simple structural principles.     

Calpactins: two distinct Ca++-regulated phospholipid- and actin-binding proteins isolated from lung and placenta

Three forms of calpactin, the 36,000 Mr Ca++-binding cytoskeletal protein, were isolated in large amounts from bovine lung and human placenta using cycles of calcium-dependent precipitation followed by solubilization with EGTA-containing buffers. Calpactin-I as a tetramer of heavy (36 kD) and light (11 kD) chains was the predominant form of calpactin isolated, however milligram amounts of the calpactin-I heavy chain monomer and calpactin-II, a related but distinct molecule, were also isolated by this method. Calpactin-II was characterized in some detail and found to bind two Ca++ ions with Kd's of 10 microM in the presence of phosphatidylserine. Both calpactin-I and -II were found to aggregate liposomes at micromolar Ca++ concentrations, suggesting that at least two phospholipid-binding sites are present on these molecules. Both calpactin monomers bind to and bundle actin filament at high (1 mM) but not low (less than 1 microM) Ca++ concentrations. Amino-terminal sequence analysis of a lower molecular mass variant of calpactin-II revealed that this protein was the previously identified human "lipocortin" molecule. Antibodies were elicited to calpactin-I and -II and the cell and subcellular distribution of each was compared. Calpactin-II was only present at high levels in tissues (lung, placenta) which contained high levels of calpactin-I. Other tissues (intestine) contained high calpactin-I and undetectable levels of calpactin-II. Double-label immunofluorescence microscopy on human fibroblasts revealed that, like calpactin-I, calpactin-II is present in a submembraneous reticular network, although the distribution of the two calpactins is not identical.     

The neuronal actin-binding proteins,neurabin I and neurabin II,recruit specific isoforms of protein phosphatase-1 catalytic subunits

Neurabins are protein phosphatase-1 (PP1) targeting subunits that are highly concentrated in dendritic spines and post-synaptic densities. Immunoprecipitation of neurabin I and neurabin II/spinophilin from rat brain extracts sedimented PP1gamma1 and PP1alpha but not PP1beta. In vitro studies showed that recombinant peptides representing central regions of neurabins also preferentially bound PP1gamma1 and PP1alpha from brain extracts and associated poorly with PP1beta. Analysis of PP1 binding to chimeric neurabins suggested that sequences flanking a conserved PP1-binding motif altered their selectivity for PP1beta and their activity as regulators of PP1 in vitro. Assays using recombinant PP1 catalytic subunits and a chimera of PP1 and protein phosphatase-2A indicated that the C-terminal sequences unique to the PP1 isoforms contributed to their recognition by neurabins. Collectively, the results from several different in vitro assays established the rank order of PP1 isoform selection by neurabins to be PP1gamma1 > PP1alpha > PP1beta. This PP1 isoform selectivity was confirmed by immunoprecipitation of neurabin I and II from brain extracts from wild type and mutant PP1gamma null mice. In the absence of PP1gamma1, both neurabins showed enhanced association with PP1alpha but not PP1beta. These studies identified some of the structural determinants in PP1 and neurabins that together contribute to preferential targeting of PP1gamma1 and PP1alpha to the mammalian synapse.     

Specific lipoxygenase isoforms accumulate in distinct regions of soybean pod walls and mark a unique cell layer

Developing seeds constitute a strong sink for the plant and rely on the turnover and mobilization of carbon and nitrogen assimilates to supply the nutrients needed for their maturation. In large part these nutrients emanate from the vegetative organs including leaves and pod walls. Vegetative lipoxygenases (VLXs) accumulate in the paraveinal mesophyll cell layer of soybean (Glycine max L.) leaves where individual isoforms are proposed to play a role(s) as active enzymes or as transient storage proteins. VLXs also are prominent proteins in soybean pod walls, representing approximately 12% of the total soluble protein. Examining the temporal, tissue, and subcellular patterns of individual VLX isoform accumulation and of lipoxygenase activity through pod wall development indicates that VLXD is the principal VLX isoform playing a role in storage in this organ. The major accumulation of VLXD occurs just prior to seed fill within the endocarp middle zone, and protein extracted from this region shows relatively low levels of lipoxygenase activity, suggesting the middle zone may act as a storage tissue. Three other VLX isoforms, VLXA, VLXB, and VLXC colocalize to the cytoplasm of a single discrete cell layer in the mesocarp. Thus, the patterns of VLX cellular and subcellular localization in pod walls suggest independent functions for these different isoforms while also serving as specific markers for a novel cell layer in the pod wall.     

Sorting of tropomyosin isoforms in synchronised NIH 3T3 fibroblasts: evidence for distinct microfilament populations

The nonmuscle actin cytoskeleton consists of multiple networks of actin microfilaments. Many of these filament systems are bound by the actin-binding protein tropomyosin (Tm). We investigated whether Tm isoforms could be cell cycle regulated during G0 and G1 phases of the cell cycle in synchronised NIH 3T3 fibroblasts. Using Tm isoform-specific antibodies, we investigated protein expression levels of specific Tms in G0 and G1 phases and whether co-expressed isoforms could be sorted into different compartments. Protein levels of Tms 1, 2, 5a, 6, from the alpha Tm(fast) and beta-Tm genes increased approximately 2-fold during mid-late G1. Tm 3 levels did not change appreciably during G1 progression. In contrast, Tm 5NM gene isoform levels (Tm 5NM-1-11) increased 2-fold at 5 h into G1 and this increase was maintained for the following 3 h. However, Tm 5NM-1 and -2 levels decreased by a factor of three during this time. Comparison of the staining of the antibodies CG3 (detects all Tm 5NM gene products), WS5/9d (detects only two Tms from the Tm 5NM gene, Tm 5NM-1 and -2) and alpha(f)9d (detects specific Tms from the alpha Tm(fast) and beta-Tm genes) antibodies revealed 3 spatially distinct microfilament systems. Tm isoforms detected by alpha(f)9d were dramatically sorted from isoforms from the Tm 5NM gene detected by CG3. Tm 5NM-1 and Tm 5NM-2 were not incorporated into stress fibres, unlike other Tm 5NM isoforms, and marked a discrete, punctate, and highly polarised compartment in NIH 3T3 fibroblasts. All microfilament systems, excluding that detected by the WS5/9d antibody, were observed to coalign into parallel stress fibres at 8 h into G1. However, Tms detected by the CG3 and alpha(f)9d antibodies were incorporated into filaments at different times indicating distinct temporal control mechanisms. Microfilaments in NIH 3T3 cells containing Tm 5NM isoforms were more resistant to cytochalasin D-mediated actin depolymerisation than filaments containing isoforms from the alpha Tm(fast) and beta-Tm genes. This suggests that Tm 5NM isoforms may be in different microfilaments to alpha Tm(fast) and beta-Tm isoforms even when present in the same stress fibre. Staining of primary mouse fibroblasts showed identical Tm sorting patterns to those seen in cultured NIH 3T3 cells. Furthermore, we demonstrate that sorting of Tms is not restricted to cultured cells and can be observed in human columnar epithelial cells in vivo. We conclude that the expression and localisation of Tm isoforms are differentially regulated in G0 and G1 phase of the cell cycle. Tms mark multiple microfilament compartments with restricted tropomyosin composition. The creation of distinct microfilament compartments by differential sorting of Tm isoforms is observable in primary fibroblasts, cultured 3T3 cells and epithelial cells in vivo.     

Opposite effects of PDGF-BB and prostaglandin E1 on cell-motility related processes are paralleled by modifications of distinct actin-binding proteins

Prostaglandin E₁ (PGE₁) lowers dermal interstitial fluid pressure (IFP) in vivo and inhibits fibroblast-mediated collagen gel contraction in vitro. PDGF-BB, in contrast, stimulates contraction and normalizes IFP lowered as a result of anaphylaxis. Human diploid AG1518 fibroblasts expressed EP2, EP3 and IP prostaglandin receptors. The inhibitory effect of PGE₁ on contraction depended on cAMP. Short-term stimulation with PDGF-BB transiently induced formation of actin-containing membrane and circular ruffles and breakdown of stress fibers. PGE₁ had no effect on stress fibers nor did it modulate the effects of PDGF-BB. PGE₁ alone or in combination with PDGF-BB inhibited initial adhesion and spreading to collagen. PDGF-BB had no effect on adhesion but stimulated cell spreading. Two-dimensional gel electrophoresis and MALDI TOF analyses of SDS/Triton X-100-soluble proteins revealed changes in migration pattern of actin-binding proteins. Interestingly, PDGF-BB and PGE₁ affected both similar and different sets of actin-binding proteins. PDGF-BB and PGE₁ did not trans-modulate their respective effects on actin-binding proteins, cytoskeletal organization or initial adhesion. Our data show that PDGF-BB stimulates actin cytoskeleton dynamics, whereas PGE₁ inhibits processes dependent on cytoskeletal motor functions. We suggest that these different activities may partly explain the contrasting effects of PGE₁ and PDGF-BB on contraction and IFP.     

Heterodimeric capping proteins constitute a highly conserved group of actin-binding proteins.

The two subunits of the heterodimeric protein cap32/34, an actin-binding protein, are encoded by separate single-copy genes. We have established the genomic structure of both genes. A sequence comparison of cap32/34 with capZ from chicken skeletal muscle and two partially known sequences from Saccharomyces cerevisiae and Xenopus laevis show that heterodimeric capping proteins belong to a highly conserved group of actin-binding proteins. This conclusion is supported by the cross-reaction of polyclonal antibodies against cap32 and cap34 with proteins from lower and higher eukaryotes. In addition, a system is presented that allows the expression of truncated cap34 polypeptides under the control of the cap34 promoter.     

The function of actin-binding proteins in pollen tube growth

Pollen tube growth is a key step in sexual reproduction of higher plants. The pollen tube is a typical example of tip-growing cells and shows a polarized cytoplasm. To develop and maintain polarized growth, pollen tubes need a carefully regulated actin cytoskeleton. It is well known that actin-binding proteins are responsible for the direct control of dynamic actin filaments and serve as a link between signal transduction pathways and dynamic actin changes in determining cellular architecture. Several of these classes have been identified in pollen tubes and their detailed characterisation is progressing rapidly. Here, we aim to survey what is known about the major actin-binding proteins that affect actin assembly and dynamics, and their higher-order organisation in pollen tube growth.