Douglas B. Murphy

G PROTEIN SIGNALING EVENTS ARE ACTIVATED AT THE LEADING EDGE OF CHEMOTACTIC CELLS

Directional sensing by eukaryotic cells does not require polarization of chemoattractant receptors. The translocation of the PH domain-containing protein CRAC in D. discoideum to binding sites on the inner face of the plasma membrane reflects activation of the G protein-linked signaling system. Increments in chemoattractant elicit a uniform response around the cell periphery. Yet when cells are exposed to a gradient, the activation occurs selectively at the stimulated edge, even in immobilized cells. We propose that such localized activation, transmitted by the recruitment of cytosolic proteins, may be a general mechanism for gradient sensing by G protein-linked chemotactic systems including those involving chemotactic cytokines in leukocytes.

Sequestration of Tubulin in Neurons in Alzheimer's disease

In Alzheimers disease (AD), a variety of populations of neurons exhibits cytoskeletal abnormalities, including neurofibrillary tangles (NFT) in perikarya, Hirano bodies in dendrites and filament-distended axons/terminals/dendrites (neurites) in senile plaques. Some nerve cells, particularly pyramidal neurons of the hippocampus, also develop Hirano bodies (paracrystalline arrays of actin) and granulovacuolar degeneration (GVD; granular inclusions in cytoplasmic vacuoles). Since abnormalities of cytoskeletal elements have been implicated in the formation of NFT, neurites and Hirano bodies, the present study was designed to determine whether GVD also may represent a type of cytoskeletal pathology. Sections of hippocampus from controls and from individuals with AD were stained by immunocytochemical methods using monoclonal and polyclonal antibodies directed against a variety of cytoskeletal antigens. Granules of GVD contained tubulin-like immunoreactivity and absorption with purified tubulin abolished staining. Other antigens were not demonstrated in granules when antibodies directed against other cytoskeletal antigens were used. The observation of sequestration of tubulin in granules is consistent with the concept that abnormalities of the neuronal cytoskeleton are an important part of the cellular pathology of AD.

Functions of tubulin isoforms

The biological significance of tubulin isotypes lies in their ability to function in different chemical and physical environments. Recent papers document the origin and distribution of several new tubulin isotypes and suggest new ways for studying their assembly and function in specialized cells.

Arrest of pigment granule motion in erythrophores by quick-freezing

We report the use of quick-freezing, as an alternative to conventional chemical fixation, to arrest the movement of pigment granules at various stages of the dispersion-aggregation cycle in Holocentrus erythrophores. During pigment aggregation, the granules in these cells move at up to 20 microns/sec, hence the structural changes underlying the movement are likely to be too fleeting to be captured faithfully by conventional aldehyde fixation. On the other hand, quick-frozen cells, when examined by freeze-etch electron microscopy, provide novel views of certain cytoplasmic components which appear to be involved in pigment granule movement, namely, fine (2- to 6-nm diameter) fibrils which link the granules to each other and to the radial array of microtubules. These fine crosslinking fibrils can be distinguished from thicker (8- to 15-nm diameter) strands of coherent granular material which pervade the cytoplasm of pigment-dispersed as well as pigment-aggregated cells. This granular matrix is removed by detergent permeabilization, after which it becomes apparent that the fine fibrils are insoluble and are distributed both within and distal to the aggregated pigment mass. The diameter of the specific fibrils does not change during pigment motion, which indicates that they are not contractile.

The pattern of MAP-2 binding on microtubules: visual enhancement of MAP attachment sites by antibody labeling and electron microscopy

We used affinity-purified rabbit antibody to hog brain microtubule-associated protein 2 (MAP-2) to examine the pattern of attachment of MAPs to microtubules purified by cycles of in vitro assembly and disassembly. Microtubules were fixed, deposited on EM grids, and labeled with antibody and protein A-gold colloid followed by negative staining. We observed that: The sites of MAP attachment were greatly enhanced by antibody binding in negatively stained preparations. The axial repeat revealed by antibody (100 +/- 5 nm) was greater than the previously reported value of 32 nm based on thin sectioning and negative staining procedures. The antibody was arranged in a broad band and revealed a helical pattern of binding. Microtubules with and without treatment with alpha-chymotrypsin to remove the projection portion of MAP-2 looked similar, suggesting that the antibody-enhanced pattern may reflect the sites of MAP attachment on microtubules. Microtubules with an increased MAP:tubulin ratio exhibited the same 100-nm periodicity.