William D. Cohen

The cytoskeletal system of nucleated erythrocytes.

Publisher Summary This chapter focuses on cytoskeleton formation and function of the marginal band (MB) of microtubules (MTs). It highlights the cytological, ultrastructural, and molecular observations on nonmammalian erythrocytes. The chapter also discusses comparative work on the cytoskeleton of primitive nucleated erythrocytes in developing mammals. All of the mature nucleated erythrocytes of nonmammalian vertebrates contain marginal bands (MBs). The MB is principally a hooplike continuous microtubule (MT) bundle located close to the plasma membrane in the plane of flattening. For erythrocytes of nonmammalian vertebrates, MB thickness in the light microscope and MT number per electron microscopic (EM) cross section are positively correlated with cell size. The MB is flexible both in situ and after isolation—that is, it can bend and twist without breaking. Tubulin is the major molecular component of MBs. Tubulin was found to constitute 1% of total cell protein, with less than one-half in the MB. The assembly of long MTs typical of MBs might be regulated by a tubulin oligomer pool, limiting the nucleation rate and accounting for at least some non-MB tubulin in the cell.

Elliptical Versus Circular Erythrocyte Marginal Bands: Isolation, Shape Conversion, and Mechanical Properties

Differentiation of nucleated erythrocytes involves transformation from spheroids to flattened discoids to mature flattened ellipsoids. The marginal band (MB) of microtubules is required for this process and continues to play a role in maintaining mature ellipsoidal cell shape. One hypothesis for MB function is that cell ellipticity is generated and maintained by asymmetric application of force across a flexible, circular MB frame by the membrane skeleton or other transverse elements. This is based on an earlier finding that isolated erythrocyte MBs are much more circular than MBs in situ. However, our present studies of salamander erythrocyte MBs isolated by a detergent-based method challenge this hypothesis. Most of these isolated MBs are initially elliptical, even though they lack transverse material (= E-MBs). They can be stabilized in that form for long periods and can be converted experimentally into the circular form (= C-MBs) by extended incubation in isolation medium or by treatment with elastase or subtilisin. We have tested an alternative hypothesis for generation and maintenance of ellipsoidal MBs, one based on intrinsic differential bending resistance and supported by construction of models. Using laser microsurgical transection to compare mechanical responses of isolated E-MBs and C-MBs, we have found their behavior to be quite different. Whereas C-MBs linearize, most E-MBs do not, instead retaining considerable curvature. These results are incompatible with the differential bending resistance hypothesis, which predicts both C-MB and E-MB linearization. However, they are consistent with a third model, in which material bound to the MB stabilizes it in the mature ellipsoidal form, and indicate that the mechanism for maintenance of MB ellipticity differs from that involved in its generation.

Cytoskeletal Events Preceding Polar Body Formation in Activated Spisula Eggs

Polar body formation is of interest both as a fundamental process in sexual reproduction and as an extreme example of unequal cytokinesis in cell biology. Eggs of the surf clam Spisula solidissima, released in the germinal vesicle stage, are readily induced to form polar bodies by activation with KCl or by fertilization. However, although Spisula eggs are utilized in current studies of centrosomes and the cell cycle (e.g., 1), polar body formation in this model system is described only in older literature (2, 3). We have examined changes occurring in major cytoskeletal elements—microtubules and F-actin—in the stages immediately preceding formation of the fi rst polar body. These stages are thought to be critical for docking of the meiotic spindle with the cell cortex, and for meiotic apparatus-cortex signaling that mediates the positioning and generation of the contractile ring. These processes occur by as yet unknown mechanisms. In this study, confocal fl uorescence microscopy was used to localize actin and tubulin relative to meiotic chromosomal stage. Ripe Spisula were obtained from the Aquatic Resources Division

In vitro morphogenesis of amphibian erythroblasts.

Non‐mammalian vertebrate erythrocytes are flattened nucleated ellipsoids containing marginal bands (MBs) of microtubules that assemble during cellular morphogenesis. Earlier work suggested that pointed erythroid cells containing pointed MBs were intermediate stages in terminal differentiation, rather than aberrant forms, but direct evidence was lacking. Here we report on morphogenesis in individual post‐cytokinetic amphibian erythroblasts in culture. Daughter cells remained adjacent in pairs, and developed pointed morphology over 1–2h in the following sequence: (a) ends opposite the cytokinetic furrow became pointed, producing a spheroidal singly‐pointed stage; (b) furrow ends usually became pointed, yielding doubly‐pointed cells; (c) furrow‐end points disappeared, producing a second singly‐pointed stage that was flattening. Over a longer term, the single points sometimes disappeared, yielding a flattened discoid. These observations support the hypothesis that pointed cells are normal intermediates in a biogenetic program in which post‐mitotic centrosomes organize MBs while occupying the singly‐pointed ends of differentiating erythroblasts.

THE TRAINING OF CELL BIOLOGISTS

In challenging members of the IFCB Education Committee to address the subject of training cell biologists, CBI Editor Denys Wheatley has posed the following questions: (1) What merits exist in the training systems in one country or region which might suitably be applied elsewhere? (2) How might the Internet, journals, and conference sessions help to address training problems? (3) What do we really mean when we say we want to train cell biologists—what is our objective? Obviously, the last question is a basic one for which answers may vary depending upon country or region. From my current U.S. perspective, the objective is also a basic one: the training of research scientists who then go on to fill any niches they so desire. This paper presents a few thoughts about maintaining and perhaps improving the quality of that process.† Consideration of the training of new researchers specializing in cell biology requires a retreat from the trees to observe the forest. As ‘cell biologists’, we deal with that level of biological organization at which the full palette of functional properties defining living systems first clearly emerges. Therefore, our students must be prepared for the study of life—and death—at its most fundamental level. Although genomics has now put reams of information about genes and their products at our disposal, the fact that cells are not just bags of genes and products has not changed. The whole remains far