Worm Breeder's Gazette 13(5): 64 (February 1, 1995)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Department of Genetics, Washington University, St. Louis, MO 63110 The C. elegans actin related protein-1 (Arp-1) shows ~50% homology to conventional actins, but is even more (~67%) similar to mammalian and chicken Act-RPV (Actin-related protein-Vertebrates). Act-RPV is a component of the multi-protein dynactin complex, which activates cytoplasmic dynein based vesicle transport along microtubules (1). Besides Act-RPV, the dynactin complex also includes actin, actin-capping protein, dynactin, and several unidentified proteins. Mutations in the gene encoding ACT-5, the S.cerevisiae homolog of Arp-1/Act-RPV, result in a defect in orientation of the spindle along the mother-bud axis during mitosis(2). This phenotype is also seen in yeast dynein heavy chain mutants (3,4), suggesting that both Act-5p (a component of yeast dynactin?) and dynein are involved in mitotic spindle orientation. In C. elegans, immunolocalization of actin and actin-capping protein during early embryogenesis show a pattern of transient structures that implicate these two proteins, and perhaps a dynactin complex, in the centrosomal-nuclear rotations in the P1 lineage (5, 6). If a dynactin complex is involved in centrosomal-nuclear rotations, one would predict co-localization of Arp-1 with actin and actin-capping protein in the transient structures during embryogenesis. To test this prediction, we have stained C. elegans embryos with antibodies raised against the yeast Act-5p, (a kind gift from Muhua Li et al), that recognize C. elegans Arp-1 as a GST-fusion protein made in E.coli. 2-D immunoblots of mixed stage N2 extracts with these antibodies show cross-reactivity to conventional actin, but there is no significant cross-reactivity to cytoplasmic or muscle actin in intact, fixed embryos (see below). We are currently using these antibodies to stain embryos homozygous for the deficiency jDf4 (right end of chromosome II) that deletes arp-1, to test the specificity of staining. Early C. elegans embryos were stained with these antibodies and, for reference, counterstained with antibodies to various cytoskeletal proteins. A summary of localization of Arp-1 follows: (I) Arp-1 is present at the Cell Division Remnants (CDRs): Arp-1 accumulates at CDRs immediately after each cell division as a prominent dot between sister cells in all cell lineages. CDRs are small ring shaped structures between sister cells that also stain with phalloidin, a reagent that binds filamentous actin (5). Hird & White have previously shown that kinesin localizes at a cortical site between AB and P1 (7). We find that this staining also co-localizes with the CDR. Thus, the CDR is marked by staining with phalloidin, anti-Arp-1, and anti-kinesin. In contrast to these three, which appear immediately upon cell division, actin and actin- capping protein appear later and only transiently during a time window that overlaps with centrosome-nuclear rotation. Even though actin and actin-capping protein appear later, they do co-localize with Arp-1 at the CDR. Since Arp-1 appears at CDRs adjacent to cells that do or do not display centrosome-nuclear rotation, it suggests that while Arp-1 may play a role in rotation, it more likely participates in events that are common to CDRs in both AB and P1 lineages. (II) CDR internalization?: Staining with phalloidin, anti-Arp-1, and anti- kinesin revealed that all three components found at the CDRs internalize and co-localize into the interior of specific cells of the P1 lineage. For example, at some point during the P1 division, the staining (with any of the three markers) disappears from the surface between AB and P1 and appears in the interior of P1 as a prominent dot or a ring, the latter seen more frequently in case of phalloidin staining. The staining appears mostly within a few microns of the anterior centrosome, but rarely coincides with the position of the anterior centrosome. Since staining is never found both in the interior and on the surface, it suggests that the structure marked by these antibodies/phalloidin might migrate inside the cell, or alternatively, it may disassemble at the surface and reassemble at an interior site. The co-localization of all three markers of CDR in the interior suggests the possibility that the entire CDR is internalized. The staining pattern is more complex at the 4 to 6 cell stages and later. In brief, new sites of staining appear at the CDR between EMS and P2, and between ABa and ABp. When ABa and ABp are in mitosis or later, the CDR staining between ABa and ABp disappears, and a new internal staining appears inside EMS. Similarly, internal staining also appears inside MS, P2, and C, but the characterization is incomplete. (1) Lees-Miller et al, Nature 359 (1992) 244. (2) Muhua Li et al, Cell 78 (1994) 669. (3) Eshel et al, PNAS 90 (1993) 11172. (4) Li et al, PNAS 90 (1993) 10096. (5) Waddle J. A. et al, Mol. Biol. Cell 4 (1993) 907-917. (6) Hyman A. and White J.J., J.Cell Biol. 105 (1987) 2123. (7) Hird, S. and White, J. WBG Vol 12 (2) pp 91 (1992).