Worm Breeder's Gazette 8(1): 33
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.
At the May, 1983 Worm Meetings, I described 2-nm filaments found in the pseudopods of crawling sperm. Recently, much better images of these filaments have been obtained by fixing intact crawling cells on nickel grids, positive staining with uranyl acetate, and critical- point drying. Sperm flatten their pseudopods when they crawl ( thickness ~1-2 m) so that the edges of the pseudopod of whole cells can be studied in a conventional electron microscope operated at 100 KV. These intact cells contain an impressive array of 2-nm filaments surrounded by the amorphous, granular pseudopod cytoplasm. The filaments are relatively short ( 100 nm) and are most oriented nearly parallel to the long axis of the cell. I often find several filaments of about the same length lying parallel to one another but have not seen any interconnections between them. Many of the filaments at the very edge of the pseudopod abut the cytoplasmic face of the plasma membrane. One sidelight of this work has been the continued, consistent failure to find filamentous actin. Sperm clearly lack F- actin and, therefore, must use a different mechanism to crawl. The polypeptide that makes up the 2-nm filaments has not yet been identified. Major sperm protein, described by Mike Klass, remains the leading candidate based on its abundance, its localization in the pseudopod, and its colocalization (by immunofluorescence) with filamentous structures in fibrous bodies of spermatocytes (Ward and Klass, 1982, Dev. Biol. 92: 203-208). I have been trying to immunolabel the 2-nm filaments with an antibody to MSP (we have both a polyclonal rabbit anti-MSP, obtained from Sam Ward, and a new monoclonal anti-MSP generated in our lab). This approach has been hindered by my inability to remove the granular cytoplasm surrounding the filaments without extracting the filaments as well. Recently, I have found that 10-30 min fixation in formaldehyde followed by extraction in Triton X-100 lyses the plasma membrane and removes much of the pseudopod cytoplasm. The pseudopod remnant consists of a honeycomb-like arrangement of cytoplasm which contains numerous 2-nm filaments. These filaments may be sufficiently freed of surrounding cytoplasm to label them convincingly with ferritin- or gold-conjugated antibodies.