Alice B. Fulton

ELECTRON MICROSCOPY OF A CONTRACTILE-VACUOLE MUTANT OF CHLAMYDOMONAS MOEWUSII (CHLOROPHYTA) DEFECTIVE IN THE LATE STAGES OF DIASTOLE1

Electron microscopy of a “vacuole‐less” mutant of Chlamydomonas moewusii Gerloff revealed the presence of small anterior vacuoles. These vacuoles behaved like contractile vacuoles in wild‐type cells, but they were apparently unable to complete diastole and discharge their contents. When wild‐type and mutant cells were incubated in hypertonic medium, small coated vacuoles persisted in the region where contractile vacuoles form. When these cells were transferred to hypotonic medium, the vacuoles appeared to fill and fuse to form larger vacuoles Shortly after the appearance of full expanded contractile vacuoles, collapsed vacuoles were observed in wild‐type cells suggesting the completion of diastole and the onset of systole. In mutant cells, the initial steps of filling and fusion to form larger vacuoles apparent interactions of vacuoles with the plasma membrane were not observed. New contractile vacuoles accumulated around the nucleus. When fusion of the contractile vacuole with the plasma membrane was blocked by EGTA, a similar accumulation of large vacuoles occurred. Our observations suggest that the contractile‐vacuole mutant of C. Moewusii produces vacuoles which can accumulate excess water as part of the mechanism of osmoregulation but which cannot complete diastole.

Treadmilling, diffusional exchange and cytoplasmic structures

SummaryMicrofilaments and microtubules exchange monomers from solution by at least two mechanisms; treadmilling and diffusional exchange. Refined kinetic analysis of both mechanisms shows that this exchange may be nonlinear under certain conditions. The two mechanisms of exchange differ in some of their predictions for the behaviour of cytoplasmic structures. Studies of assembly of cytoplasmic structuresin vivo suggest that diffusional exchange is probably predominant for steady-state structures and further suggest that additional mechanisms may be operating in the cell.

Assembly of tropomyosin isoforms into the cytoskeleton of avian muscle cells.

Tropomyosin (TM) is a component of microfilaments of most eukaryotic cells. In striated muscle, TM helps confer calcium sensitivity to the actin-myosin interaction. TM is a fibrillar, self-associating protein that binds to the extended actin filament system. We hypothesized that these structural features would permit TM to undergo assembly into the cytoskeleton during translation, or cotranslational assembly. Pulse-chase experiments with[35S]methionine and pulse experiments with [3H]puromycin followed by extraction and immunoprecipitation of TM were performed to examine the mechanism of assembly of TM into the cytoskeleton in cultured avian muscle cells. Pulse-chase experiments provide kinetic evidence for cotranslational assembly of TM in skeletal and cardiac muscle. Demonstration of a large majority of completed TM on purified skeletal muscle microfilaments after a short labeling period confirms that these kinetic data are not related to trapping of TM within the actin network of the cytoskeleton. Nascent TM peptides are demonstrated on the cytoskeleton of muscle cells after a short metabolic pulse followed by puromycin treatment to release nascent peptides from ribosomes or after labeling with [3H]puromycin. Nascent chain localization to the cytoskeleton independent of ribosomal attachment further confirms the high degree of cotranslational assembly of this protein. The extent of cotranslational assembly is similar before and after the formation of significant myofibril in myotubes, suggesting that cotranslational assembly of TM is active during contractile apparatus assembly in muscle differentiation. This is the first report where assembly mechanism has been predicted to be cotranslational based upon structural features of a cytoskeletal protein.

Interactions within the Cytoskeleton

The complex meshwork of the cytoskeleton displays three forms of interaction: structural, biochemical, and functional. As our knowledge of each form grows, it becomes increasingly clear that the cytoskeleton often behaves as a single, integrated organelle.