Worm Breeder's Gazette 8(1): 18

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.

Roles of Cytoskeletal Elements in Generation of Asymmetry and Segregation of P Granules

S. Strome, W.B. Wood

Between the time of fertilization and first cleavage of C.  elegans 
zygotes, several directed movements of cellular components, which 
reflect the asymmetry of the zygote, occur.  The zygote undergoes 
contractions of the anterior membrane and pseudocleavage.  The egg and 
sperm pronuclei migrate from opposite poles of the zygote, meet in the 
posterior hemisphere, and then move to the center.  The first mitotic 
spindle forms in the center of the zygote, but becomes asymmetric with 
respect to its position along the anterior-posterior axis and the 
behavior and morphology of its asters.
The segregation of P granules to the posterior pole of the zygote, 
which occurs concurrently with pseudocleavage and pronuclear migration,
provides one of the most striking manifestations of zygote asymmetry. 
In order to determine whether specific cytoskeletal fiber systems are 
involved in P-granule segregation and other early movements and 
whether these early events are interdependent, I have analyzed mutant 
embryos and wild-type embryos treated with microtubule (MT) and 
microfilament (MF) inhibitors.
Analysis of zyg-9(b244) embryos (described in a previous newsletter) 
and embryos treated with low levels of MT inhibitors (which partially 
phenocopy the b244 defect) demonstrate that P-granule segregation is 
not mediated by the mitotic spindle; even though spindle orientation 
is altered in mutant and treated embryos, P granules nevertheless 
coalesce and become localized in the posterior pole.  Treatment of 
zygotes with levels of MT inhibitors (colcemid, vinblastine, or 
griseofulvin) that prevent mitosis does not prevent proper P-granule 
segregation or pseudocleavage but does inhibit pronuclear migration.  
Conversely, treatment of embryos with MF inhibitors (cytochalasin D or 
B) inhibits P-granule segregation and pseudocleavage without 
preventing pronuclear migration.  In addition, the resulting spindle 
does not become asymmetric in position or in aster behavior in 
cytochalasin treated zygotes.
The results suggest that P-granule segregation does not require 
either the spindle or cytoplasmic MTs, but that this process as well 
as generation of other asymmetries do require cytoskeletal functions 
that depend on MFs.  Although MT-mediated events (pronuclear migration)
and MF-mediated events (pseudo-cleavage, P-granule segregation) are 
both normally initiated at fertilization, these two classes of 
processes can be uncoupled by drugs or mutation and therefore appear 
to be independent.  Details of these experiments will appear in Cell 
35, pp.  15-25.
In immunoprecipitation experiments designed to identify the 
molecular components of P granules, one of the anti-P-granule 
monoclonal antibodies (K76) precipitates a 37,000-dalton polypeptide 
and another anti-P-granule antibody (L416) precipitates an 83,000-
dalton polypeptide from [35S]-worm homogenates.  In gel blot 
experiments both of these antibodies bind to a ~37,000-dalton 
polypeptide in worm and egg homogenates.  However, the K76 antibody 
also binds a higher and lower molecular weight species as well.  
Further investigation of the polypeptides recognized by these 
antibodies, as well as two other anti-P-granule antibodies, is under