Centrosome dynamics in early embryos of Caenorhabditis elegans.

The early Caenorhabditis elegans embryo divides with a stereotyped pattern of cleavages to produce cells that vary in developmental potential. Differences in cleavage plane orientation arise between the anterior and posterior cells of the 2-cell embryo as a result of asymmetries in centrosome positioning. Mechanisms that position centrosomes are thought to involve interactions between microtubules and the cortex, however, these mechanisms remain poorly defined. Interestingly, in the early embryo the shape of the centrosome predicts its subsequent movement. We have used rhodamine-tubulin and live imaging techniques to study the development of asymmetries in centrosome morphology and positioning. In contrast to studies using fixed embryos, our images provide a detailed characterization of the dynamics of centrosome flattening. In addition, our observations of centrosome behavior in vivo challenge previous assumptions regarding centrosome separation by illustrating that centrosome flattening and daughter centrosome separation are distinct processes, and by revealing that nascent daughter centrosomes may become separated from the nucleus. Finally, we provide evidence that the midbody specifies a region of the cortex that directs rotational alignment of the centrosome-nucleus complex and that the process is likely to involve multiple interactions between microtubules and the cortex; the process of alignment involves oscillations and overshoots, suggesting a multiplicity of cortical sites that interact with microtubules.

Localization of the myomodulin-like immunoreactivity in the leech CNS.

The distribution of myomodulinlike immunoreactivity in the leech CNS was determined using an antiserum raised against Aplysia myomodulin. Segmental ganglia contained approximately 60 immunoreactive neurons. In addition, numerous fibers containing immunoreactive varicosities were found throughout the neuropil. Using a combination of Lucifer Yellow injections and immunocytochemistry, we identified neurons including the anterior Pagodas (AP), annulus erector (AE), motor neurons, Leydig, longitudinal muscle motoneurons (L), S cells, and coupling interneurons, all of which are active during the touch-elicited shortening reflex. FMRF-amide-like immunoreactivity in three of these cells (L, AP, and AE) was previously demonstrated. Specific staining for myomodulin was abolished by preadsorption of the antiserum with synthetic myomodulin, but not with FMRF-amide. These results suggest a potential role for myomodulin in both intrinsic and extrinsic modulation of the leech touch-elicited shortening reflex. Further, it is possible that several neurons mediating this reflex contain multiple neuromodulatory peptides.

The dynein genes of Paramecium tetraurelia. Sequences adjacent to the catalytic P-loop identify cytoplasmic and axonemal heavy chain isoforms.

Paramecium tetraurelia is a unicellular organism that utilizes both axonemal and cytoplasmic dyneins. The highly conserved region containing the catalytic P-loop of the dynein heavy chain was amplified by RNA-directed polymerase chain reaction. Eight different P-loop-containing cDNA fragments were cloned. Southern hybridization analysis indicated that each fragment corresponds to a separate dynein gene and that there are at least 12 dynein heavy chain genes expressed in Paramecium. Seven of the eight cloned contain sequence motif A, which is found in axonemal dyneins, and one contains sequence motif B, which is found in the dyneins from cell types that do not have cilia or flagella. Two of the Paramecium dynein genes were further investigated: DHC-6 which contains motif A, and DHC-8 which contains motif B. Additional sequencing of the central portions of these genes showed that DHC-6 most closely matches sea urchin ciliary beta heavy chain and DHC-8 is similar to the cytoplasmic dynein from Dictyostelium. Deciliation of the cells resulted in a substantial increase in the steady state concentration of DHC-6 mRNA but only a small change in DHC-8 mRNA. Antisera were produced against synthetic peptides derived from sequence motifs A and B. Competitive solid-phase binding assays demonstrated that each antiserum was peptide-specific. In western blots, the antiserum to motif A reacted with both ciliary and cytoplasmic dyneins. In contrast, the antiserum to motif B reacted with the cytoplasmic dyneins of Paramecium and bovine brain but did not react with ciliary dynein.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of phosphorylated microtubule-associated protein 1B during neurite outgrowth in PC12 cells.

The functional significance of microtubule-associated protein 1B (MAP1B) phosphorylation during neuronal differentiation is unknown. In the present study we examined the hypothesis that the phosphorylation of MAP1B is required for neurite outgrowth. We reasoned that if MAP1B phosphorylation was important for neurite outgrowth then the intracellular distribution of phosphorylated MAP1B might exist as a discrete subset of the pattern for total MAP1B. We utilized a monoclonal antibody (mAb 7-1.1) that specifically recognizes a phosphorylated epitope on MAP1B and a polyclonal antiserum that recognizes all MAP1B protein to compare the distributions of phosphorylated and total MAP1B during neurite outgrowth. Phosphorylated MAP1B progressively accumulated in both the soluble and cytoskeletal fractions of differentiating cells. Similar proportions of total and phosphorylated MAP1B were associated with the cytoskeletons of differentiating PC12 cells. Within individual cells, phosphorylated MAP1B, in comparison with total MAP1B, was not limited to a particular intracellular domain. Phosphorylated MAP1B was present in both neurites and cell bodies. It was associated with fibrillar microtubules in neurites and growth cones, but it appeared nonfibrillar within cell bodies. In some cells that differentiated rapidly, there was little phosphorylated MAP1B in the early neurites despite the presence of extensive microtubules. In addition, although phosphorylated MAP1B increased in populations of mature PC12 cell cultures, increases in phosphorylated MAP1B did not always correlate with neurite outgrowth in individual cells. These results suggest that the phosphorylated isoform of MAP1B recognized by mAb 7-1.1 may not be required for neurite outgrowth.