Actin Isoforms

The functional role of actin in smooth muscle in active shortening and force development is widely accepted. However, it is also well known that actin filaments play multiple roles in non-muscle cells, where they are responsible for the maintenance of cell shape and organization as well as motility (Stossel 1993). There is evidence from a number of laboratories that actin filamants of different isoforms may be functionally specialized in smooth muscle ceEs and localized to different physical domains within the cell (Lehman et al 1987, North et al 1994b, Small 1995). 

At least six different isoforms of actin hav been identified in vertebrate tissues, each encoded by a different gene (Vanderkerckhove and Weber 1978, Reddy et al 1990). Four of these isoforms have been identified in the smooth muscle tissues of warm-blooded vertebrates a and y smooth muscle actin, and p and y non-muscle actin (Kabsch and Vanderkerckhove 1992). These actin variants have approximately 95% amino acid sequence homology, and differ primarily in their N-terminal sequence (Pollard and Cooper 1986). 

The expression of the different actin genes is tissue specific. For example, the expression of y-actin is highest in visceral smooth muscles and the expression of a-actin is highest in vascular smooth muscles (Fatigati and Murphy 1984, Hartshorne 1987). Some smooth muscle tissues contain a mixture of both a and y isoforms others express one or the other exclusively (Hartshorne 1987). The p or cytoskeletal isoform of actin appears to be a significant component of all smooth muscle tissues. No functional differences among these isoforms or the skeletal muscle isoform have been discerned with respect to the activation of myosin ATPase, actin filament 

---

Just I, Wille M, Chaponnier C et al. (1993b) Gelsolin-actin complex is target for ADP-ribosylation by Clostridium botulinum C2 toxin in intact human neutrophils. In Eur. J. Pharmacol. Mol. Pharmacol. 246 293-7 Kiefer A, Lerner M, Sehr P et al. (1996) Depolymerization of F-actin by microinjection of ADP-ribosylated skeletal muscle G-actin in PtK2 cells in the absence of the ADP-ribosylating toxin. In A/ted. Microbiol. Immunol. 184 175-80 Mauss S, Chaponnier C, Just I et al. (1990) ADP-ribosylation of actin isoforms by Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin. In Eur. J. Biochem. 194 237-41... 

Skeletal muscle a-actin is to purify easily in large amounts and is stable for several weeks when stored at 4°C. In contrast, non-muscle actin (P- and y-isoforms) is difficult to prepare and stable only for a few days. Therefore, skeletal muscle actin is the preferred isoform to study functions of G-actin. Skeletal muscle a-actin, however, is not a substrate for C2 toxin. In this case, C. perfringens iota toxin (enzyme component ia) has to be used, as this modifies all actin isoforms. To study the influence of ADP-ribosylation on the properties (e.g., ATPase activity, interactions with actin-binding proteins (Geipel et al., 1989 Geipel ef al., 1990)) of isolated skeletal muscle actin, the following protocol is used. 

CC - - MISCELLANEOUS IN VERTEBRATES 3 MAIN GROUPS OF ACTIN ISOFORMS,... 

Schurch W, Skalli O, Seemayer TA, et al. Intermediate filament proteins and actin isoforms as markers for soft tissue tumor differentiation and origin. I. Smooth muscle tumors. Am J Pathol. 1987 128 91-103. 

Cintorino M, Vindigni C, DelVecchio MT, et al. Expression of actin isoforms and intermediate filament proteins in childhood orbital rhabdomyosarcomas. J Submicrosc Cytol Pathol. 1989 21 409-419. 

Sarcomatoid melanomas are consistently SIOOP positive, but only 3% to 10% can be labeled for other, more specific melanocytic determinants (Fig. 7.16). °420,121,123,192 Because a proportion of such lesions becomes transmogrified into spindle-cell proliferations with divergent phenotypes, immunoreactivity for CD56, CD57, nerve growth factor receptor, desmin, and actin isoforms is potentially observed in them (Figs. 

Schuler, H., U. Lindberg, C. E. Schutt, and R. Karlsson (2000). Thermal unfolding of G-actin monitored with the DNase 1-inhibition assay stabilities of actin isoforms. Eur. J. Biochem. 267, 476-486. 

The various actin isoforms exhibit minor sequence differences but generally perform different functions. 

Actin is a highly conserved protein, and actin variants display approximately 95% amino acid sequence homology (Pollard and Cooper, 1986). In vertebrate tissues, six different actin isoforms have been identified on the basis of electrophoretic mobility and sequence data, and each is encoded by a distinct gene (Vandekerckhove and Weber, 1978 Reddy et al., 1990). a, p, and 7 species can be easily separated by isoelectric focusing the a-isoform is the most acidic and the 7-isoform the least (Vandekerckhove and Weber, 1979a). These variants can be further subdivided on the basis of their amino acid sequence and include three a-isoforms (a-skeletal, a-cardiac, and a-vascular), two 7-isoforms (7-enteric and 7-cytoplasmic), and one p-isoform (p-cytoplasmic). 

Table I illustrates the variability of the amino-terminal residues of several different actin isoforms. Mutagenesis in this region can result in partial or complete inhibition of F-actin motility (Sutoh, 1993), consistent with the site being a likely initial target for myosin during the cross-bridge cycle (Rayment et al., 1993b). In fact, numerous studies also have identified several domains of the actin molecule representing clusters of amino acids specifically involved in monomer-monomer interactions (Holmes et al., 1990 Hennessey et al., 1993 Khaitlina et al., 1993 Labbe et al., 1994), actin-myosin interactions (Holmes and Kabsch, 1991 Hennessey et al., 1993 Schroder et al.,...

What is the reason for actin isoform diversity in smooth muscle ... 

How is the pattern of appearance of actin isoforms during smooth muscle development related to cell function ... 

How are actin isoforms segregated in smooth muscle cells ... 

Are subtle differences in actin isoform structure related to functional differences ... 

Actin occurs in multiple isoforms within individual smooth muscle cells. All of these isoforms are capable of forming filamentous actin that can interact with myosin to generate force. Although the functional importance of the different actin isoforms is presently unclear, there is evidence to suggest that they may serve to "customize actin filaments to serve different functional roles within the cell by determining its interactions with different binding proteins. The first part of this section will review the molecular structure of the thin filament. The structure of actin and the relationship to the other protein constituents of the thin filament to actin... 

Fig. 5. Schematic representation of components of the thin filament of smooth muscle. Although calponin and caldesmon are both shown as components of the same filament, there is evidence that they may selectively associate with different actin isoforms in different populations of thin filaments...

Actin isoform specific localization of thin filament proteins... 

The distribution of actin and tropomyosin are generally agreed to be highly correlated throughout the cytoplasm, and in native thin filaments, tropomyosin appears to be bound to all actin filament isoforms (Mabuchi et al 1996, Small et al 1986). However, there is evidence from a number of laboratories that different actin-binding proteins associate selectively with different actin isoforms and localize to different functional domains within smooth muscle cells. 

North AJ, Gimona M, Lando Z, Small JV (1994b) Actin isoform compartments in chicken gizzard smooth muscle cells. J Cell Sci 107 445455 OBrien EJ, Bennett PM, Hanson J (1971) Optical diffraction studies of myofibrillar structure. Philos Trans R Soc Lond B Biol Sci 261 201208 Pavalko FM, Adam LP, Wu MF, Walker TL, Gunst SJ (1995) Phosphorylation of dense-plaque proteins talin and paxUlin during tracheal smooth musde contraction. Am J Physiol 268 C56371... 

North AJ, Gimona M, Lando Z, Small JV (1994b) Actin isoform compartments in chicken gizzard smooth muscle cells. J Cell Sci 107 445-455... 

Smooth muscle-type actin isoforms Six isoforms of actin are expressed in mammalian SM tissues that are generated by distinct genes [79] and show an highly conserved amino acid sequences across species. The expression of actin isoforms is developmen-tally regulated in a temporal-spatial manner [80-83]. Fully differentiated SMC contain a- (mainly in vascular tissue) and y-(mainly in the enteric structures) actins and trace amounts of (3- and y-cytoplasmic actin isoforms [79,84-87]. The SM a-isoform is expressed also in striated muscle [88], and the SM y-isoform in post-meiotic sperm [89], and the increased SM y-actin content in hypertrophied bladder SM [86,90]. The unique intracellular distribution of actin filamentous networks [91-93] and the decreased SM a-actin content in proliferating vascular SMC ([94] see Section 6) point to an isoform diversification based on specific functional requirements. 

Heterogeneity of SM-type a-actin isoform distribution, as determined by a sequence specific monoclonal antibody [189], in vascular SM tissue is especially evident in newborn rats where a population of SMC is negative for this marker [80,190]. Apparently,A comparison of the spatiotemporal distribution of vimentin/desmin/actin during development and adult is still lacking. 

Blank RS, Owens GK (1990) Platelet-derived growth factor regulates actin isoform expression and growth state in cultured rat aortic smooth muscle cells. J Cell Physiol 142 635-642... 

Desmoulidre A, Rubbia-Brandt L, Gabbiani G (1991) Modulation of actin isoform expression in cultured arterial smooth muscle cells by heparin and culture conditions. Arterioscler Thromb 11 244-253... 

---