Ron P. Weinberger

SPECIFIC ISOFORMS OF ACTIN-BINDING PROTEINS ON DISTINCT POPULATIONS OF GOLGI-DERIVED VESICLES

Golgi membranes and Golgi-derived vesicles are associated with multiple cytoskeletal proteins and motors, the diversity and distribution of which have not yet been defined. Carrier vesicles were separated from Golgi membranes, using an in vitro budding assay, and different populations of vesicles were separated using sucrose density gradients. Three main populations of vesicles labeled with β-COP, γ-adaptin, or p200/myosin II were separated and analyzed for the presence of actin/actin-binding proteins. β-Actin was bound to Golgi cisternae and to all populations of newly budded vesicles. Centractin was selectively associated with vesicles co-distributing with β-COP-vesicles, while p200/myosin II (non-muscle myosin IIA) and non-muscle myosin IIB were found on different vesicle populations. Isoforms of the Tm5 tropomyosins were found on selected Golgi-derived vesicles, while other Tm isoforms did not colocalize with Tm5 indicating the association of specialized actin filaments with Golgi-derived vesicles. Golgi-derived vesicles were shown to bind to F-actin polymerized from cytosol with Jasplakinolide. Thus, newly budded, coated vesicles derived from Golgi membranes can bind to actin and are customized for differential interactions with microfilaments by the presence of selective arrays of actin-binding proteins.

Tropomyosin Localization Reveals Distinct Populations of Microfilaments in Neurites and Growth Cones

The functional and structural differences between neurites and growth cones suggests the possibility that distinct microfilament populations may exist in each domain. Tropomyosins are integral components of the actin-based microfilament system. Using antibodies which detect three different sets of tropomyosin isoforms, we found that the vast majority of tropomyosin was found in a microfilament-enriched fraction of cultured cortical neurons, therefore enabling us to use the antisera to evaluate compositional differences in neuritic and growth cone microfilaments. An antibody which reacts with all known nonmuscle isoforms of the alpha Tms gene (Tm5NM1-4) stains both neurites and growth cones, whereas a second antibody against the isoform subset, Tm5NM1-2, reacts only with the neurite. A third antibody which reacts with the Tm5a/5b isoforms encoded by a separate gene from alpha Tms was strongly reactive with both neurites and growth cones in 16-h cultures but only with the neurite shaft in 40-h cultures. Treatment of neurons with cytochalasin B allowed neuritic Tm5NM1-2 to spread into growth cones. Removal of the drug resulted in the disappearance of Tm5NM1-2 from the growth cone, indicating that isoform segregation is an active process dependent on intact microfilaments. Treatment of 40-h cultures with nocodazole resulted in the removal of Tm5NM1-2 from the neurite whereas Tm5a/5b now spread back into the growth cone. We conclude that the organization of Tm5NM1-2 and Tm5a/5b in the neurite is at least partially dependent on microtubule integrity. These results indicate that tropomyosin isoforms Tm5NM1-2, Tm5NM3-4, and Tm5a/5b mark three distinct populations of actin filaments in neurites and growth cones. Further, the composition of microfilaments differs between neurites and growth cones and is subject to temporal regulation.

CREATING INTRACELLULAR STRUCTURAL DOMAINS : SPATIAL SEGREGATION OF ACTIN AND TROPOMYOSIN ISOFORMS IN NEURONS

Actin microfilaments play a direct role in a variety of cell processes. Distinct populations of microfilaments are associated with different cellular compartments, such as growth cones, filipodia, stress fibers, and lamellipodia. It is becoming clear that these different populations are often composed of different isoforms of the two core microfilament components, actin and tropomyosin. This is particularly true in neurons, where actin and tropomyosin isoforms are segregated into different intracellular compartments which correspond to functionally distinct regions of the neuron. Developmental regulation of this isoform sorting suggests a specific role for some isoforms in growth and for others in stabilization of neuronal structure. This provides a mechanism by which a neuron can create and independently regulate intracellular domains composed of microfilaments with different functional properties. BioEssays 20:892–900, 1998.

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.

Intracellular Localization of Tropomyosin mRNA and Protein Is Associated with Development of Neuronal Polarity

Neuronal differentiation involves extensive rearrangement of the cytoskeleton, including the actin-based microfilament system, and establishment of molecular compartments within the neuron. The intracellular distribution of tropomyosin (Tm) mRNA in vivo and in vitro has been examined and correlated with protein targetting. The mRNAs encoding two Tm isoforms were found to be differentially localized in developing neurons. Tm-5 mRNA is localized to the axonal pole of differentiating embryonic rat neurons, in contrast to TmBr-2 mRNA distribution throughout the cell body. Tm-5 mRNA is transported into the axon of differentiating primary cultured neurons. This mRNA localization is developmentally regulated and correlates with the targeting of Tm-5 protein to growing axons. Tm-5 colocalizes with a subset of neuronal microfilaments associated with the initiation and maintenance of outgrowth. The segregation of Tm-5 is the earliest known marker of neuronal polarity and may play a role in the establishment of polarity.

Structural Compartments within Neurons: Developmentally Regulated Organization of Microfilament Isoform mRNA and Protein

The microfilament system is thought to be a crucial cytoskeletal component regulating development and mature function of neurons. The intracellular distribution of the microfilament isoform components, actin and tropomyosin (Tm), in neurons primarily in vivo, has been investigated at both the mRNA and the protein level using isoform specific riboprobes and antibodies. Our in vivo and in vitro studies have identified at least six neuronal compartments based on microfilament isoform mRNA localization: the developing soma, the mature soma, growth cone, developing axon hillock/proximal axon, mature somatodendritic and mature axonal pole soma. Protein localization patterns revealed that the isoforms were frequently distributed over a wider area than their respective mRNAs, suggesting that isoform specific patterns of mRNA targeting may influence, but do not absolutely determine, microfilament isoform location. Tm4 and Tm5 showed identical mRNA targeting in the developing neuron but distinct protein localization patterns. We suggest that in this instance mRNA location may best be viewed as a regulated site of synthesis and assembly, rather than a regulator of protein localization per se. In addition, Tm5 and beta-actin mRNA and protein locations were developmentally regulated, suggesting the possibility that environmental signals modulate targeting of specific mRNAs and their proteins. Thus, developmentally regulated mRNA localization and positional translation may act in concert with protein transport to regulate neuronal microfilament composition and consequently neuronal structure.

Sorting of a nonmuscle tropomyosin to a novel cytoskeletal compartment in skeletal muscle results in muscular dystrophy

Tropomyosin (Tm) is a key component of the actin cytoskeleton and >40 isoforms have been described in mammals. In addition to the isoforms in the sarcomere, we now report the existence of two nonsarcomeric (NS) isoforms in skeletal muscle. These isoforms are excluded from the thin filament of the sarcomere and are localized to a novel Z-line adjacent structure. Immunostained cross sections indicate that one Tm defines a Z-line adjacent structure common to all myofibers, whereas the second Tm defines a spatially distinct structure unique to muscles that undergo chronic or repetitive contractions. When a Tm (Tm3) that is normally absent from muscle was expressed in mice it became associated with the Z-line adjacent structure. These mice display a muscular dystrophy and ragged-red fiber phenotype, suggestive of disruption of the membrane-associated cytoskeletal network. Our findings raise the possibility that mutations in these tropomyosin and these structures may underpin these types of myopathies.

Distinct localizations of tropomyosin isoforms in LLC‐PK1 epithelial cells suggests specialized function at cell–cell adhesions

At least eight nonmuscle, nonbrain tropomyosin isoforms have been described. We used antibodies, microinjection, and transfection to characterize their expression and localization in LLC-PK1 kidney epithelial cells and compared them with other cells. Similar to primary enterocytes, LLC-PK1 cells exhibited predominantly TM-1 and TM-3 of the high-molecular-weight (HMW) isoforms; TM-5 and TM-5b of the low-molecular-weight (LMW) isoforms. Neither TM-4 nor TM-5a was detectable in the LLC-PKI cells. Immunofluorescence studies revealed that HMW isoforms were localized only on stress fibers, not adhesion belts, whereas the adhesion belts were stained by LMW isoform antibodies. When exogenous proteins are introduced either by transfection or microinjection, the HMW isoforms do not incorporate into the adhesion belt, whereas the LMW isoforms can incorporate into the stress fibers, thus indicating there are different mechanisms at work for the selective localization. Temporal changes in the microfilament system of the LLC-PK1 cells were studied during differentiation in culture as defined by spectrin expression and F-actin architecture. Western blot analysis indicated that TM-5b is only expressed in the LLC-PK1 cells after a certain degree of maturation in culture, which suggests isoform switching after the cell-cell contacts are developed. Collectively these results demonstrate that epithelial cells express a complex pattern of TM isoforms, which exhibit differential localizations within the cells and different patterns of expression depending on their origin and stage of differentiation. The implication of differential localization of TM isoforms on their specific functions is discussed.

Splicing of two internal and four carboxyl-terminal alternative exons in nonmuscle tropomyosin 5 pre-mRNA is independently regulated during development.

Four nonmuscle tropomyosin isoforms have been reported to be produced from the rat Tm5 gene by alternative splicing (Beisel, K. W., and Kennedy, J. E. (1994) Gene (Amst.) 145, 251–256). In order to detect additional isoforms that might be expressed from that gene, we used reverse transcriptase-polymerase chain reaction assays and evaluated the presence of all product combinations of two alternative internal exons (6a and 6b) and four carboxyl-terminal exons (9a, 9b, 9c, and 9d) in developing and adult rat brain. We identified five different combinations for exon 9 (9a + 9b, 9a + 9c, 9a + 9d, 9c, and 9d), and the exon combinations 9a + 9c and 9a + 9d were previously unreported. Each of these combinations existed with both exon 6a and exon 6b. Thus, the rat brain generates at least 10 different isoforms from the Tm5 gene. Northern blot hybridization with alternative exon-specific probes revealed that these isoforms were also expressed in a number of different adult rat tissues, although some exons are preferentially expressed in particular tissues. Studies of regulation of the 10 different Tm5 isoform mRNAs during rat brain development indicated that no two isoforms are coordinately accumulated. Furthermore, there is a developmental switch in the use of exon 6a to exon 6b from embryonic to adult isoforms. TM5 protein isoforms show a differential localization in the adult cerebellum.

Actin and tropomyosin isoforms in morphogenesis.

Abstract The major components of the actin microfilament system, actin and tropomyosin (Tm), are encoded by multigene families. There are at least 6 actin and over 20 Tm isoforms in mammals. The observation that isoforms are expressed in a tissue-specific manner has encouraged the hypothesis that they contribute to the formation of cell type-specific structures. Recent studies have indicated that certain specific isoforms do play unique structural roles. One nonmuscle actin isoform, β, is implicated in the regulation of cell spreading and membrane organisation. The intracellular location of β- actin mRNA has been shown to be regulated by growth factor stimulation of signal transduction pathways. Actin isoforms have also been shown to differ in their contractile properties in both muscle and non-muscle cells. Tropomyosins have been found to show isoform specific regulation in response to cell transformation. This has correlated with the view that some isoforms of tropomyosin promote filament stability whereas others are associated with more dynamic structures. Neuronal development and maturation are accompanied by dynamic spatial sorting of tropomyosin isoforms into different cellular compartments. It is now apparent that isoforms of these proteins perform different structural tasks. The challenge is now to link the significance of spatial sorting to the different physicochemical properties of these isoforms.