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.


Paresh V. Shrimankar, Lawrence A. Schriefer, and Robert H. Waterston

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).