alpha Spectrin

DEVELOPMENTAL BIOLOGY (part 1/2)

Embryonic

The early embryonic divisions of Drosophila are characterized by rapid, synchronized changes of the nuclei and surrounding cytoskeleton. These changes are carried out by two separately organized systems -- one based on DNA and another on centrosomes. In the nuclear system, DNA is sufficient to cause assembly of nuclear lamina and the formation of nuclear membrane with pore structures. Free centrosomes are correlated with the formation of microtubule, microfilament and spectrin networks in the absence of nuclei. The morphology of the cytoskeleton, associated with the free centrosomes, cycles in response to the embryonic cell cycle cues. These observations suggest that the centrosomes may be responsible for the organization of this extensive cytoskeleton. The early divisions may therefore result from the independent cycling of the two systems (the nucleus and the surrounding cytoskeleton) that respond separately to the mitotic cues in the embryo and function together to give the synchronized early divisions. The Drosophila embryo has an "intermediate" mitotic system in which the nuclear membrane does not break down completely during mitosis. It is speculated that the principles of cytoskeleton organization in this system may be different from those of the Xenopus "open" mitotic system (Yasuda, 1991).

The distribution of alpha Spectrin in Drosophila embryos was determined by immunofluorescence using affinity-purified polyclonal or monoclonal antibodies. During early development, spectrin is concentrated near the inner surface of the plasma membrane, in cytoplasmic islands around the syncytial nuclei, and, at lower concentrations, throughout the remainder of the cytoplasm of preblastoderm embryos. As embryogenesis proceeds, the distribution of spectrin shifts with the migrating nuclei toward the embryo surface so that, by nuclear cycle 9, a larger proportion of the spectrin is concentrated near the plasma membrane. During nuclear cycles 9 and 10, as the nuclei reach the cell surface, the plasma membrane-associated spectrin becomes concentrated into caps above the somatic nuclei. Concurrent with the mitotic events of the syncytial blastoderm period, the spectrin caps elongate at interphase and prophase, and divide as metaphase and anaphase progress. During cellularization, the regions of spectrin concentration appear to shift: spectrin increases near the growing furrow canal and concomitantly increases at the embryo surface. In the final phase of furrow growth, the shift in spectrin concentration is reversed: spectrin decreases near the furrow canal and concomitantly increases at the embryo surface. In gastrulae, spectrin accumulates near the embryo surface, especially at the forming amnioproctodeal invagination and cephalic furrow. During the germband elongation stage, the total amount of spectrin in the embryo increases significantly and becomes uniformly distributed at the plasma membrane of almost all cell types. The highest levels of spectrin are in the respiratory tract cells; the lowest levels are in parts of the forming gut. The spatial and temporal changes in spectrin localization suggest that this protein plays a role in stabilizing rather than initiating changes in structural organization in the embryo (Pasacreta, 1989).

Larval

The apical subcellular distribution of ß_H/Karst in both the wing and eye imaginal discs colocalizes with Shotgun (DE-cadherin) at the adherens junction, as it does in embryonic epithelia. This association appears intimate: in regions of the eye disc where Shotgun is more abundant, ß_H is also more prominent. The mutual exclusivity between ß_H and the conventional ß-spectrin isoform is the norm and may be important for cell polarization. While ß_H is localized to the adherens junction in imaginal disc epithelia, alpha-spectrin (presumably partnered by ß-spectrin) extends into the basolateral domain. This is consistent with the apical restriction of ß_H in other epithelia and the consistent restriction of ß-spectrin to the basolateral membrane. To establish such a situation, different proteins must recruit the different spectrins to each domain. In the fly, conventional ß-spectrin is recruited to the membrane by ankyrin, while ß_H does not colocalize with ankyrin and has no conserved ankyrin-binding site, suggesting that its interaction with the membrane is not ankyrin dependent. Binding to ankyrin could thus be used to specifically recruit ß-spectrin to the basolateral membrane. Presumably the reciprocal situation with some as yet unidentified receptor for ß_H results in its apical restriction. This mutually exclusive targeting makes it unlikely that the variable nature of the karst mutation is due to partial redundancy of the two ß-isoforms (Thomas 1998 and references).

Adult

Fusome-specific antibodies were used to characterize the early stages of cyst formation. During the first cystoblast division, a spherical mass of fusome material (the "spectrosome") is associated with only one pole of the mitotic spindle, revealing that this division is asymmetric. During the subsequent three divisions, the growing fusome always associates with the pole of each mitotic spindle that remains in the mother cell, and only extends through the newly formed ring canals after each division is completed. These observations suggest that fusomes help establish a system of directional transport between cystocytes that underlies oocyte determination (Lin, 1995).

In the Drosophila ovary, membrane skeletal proteins such as the adducin-like Hts protein(s), Spectrin, and Ankyrin are found in the spectrosome, an organelle in germline stem cells (GSC) and their differentiated daughter cells (cystoblasts). These proteins are also components of the fusome, a cytoplasmic structure that spans the cystoblast's progeny that develop to form a germline cyst consisting of 15 nurse cells and an oocyte. Spectrosomes and fusomes are associated with one pole of spindles during mitosis and are implicated in cyst formation and oocyte differentiation. The asymmetric behavior of the spectrosome persists throughout the cell cycle of GSC. Eliminating the spectrosome by the htsl mutation leads to randomized spindle orientation, suggesting that the spectrosome anchors the spindle to ensure the asymmetry of GSC division; eliminating the fusome in developing cysts results in defective spindles and randomized spindle orientation as well as asynchronous and reduced cystocyte divisions. These observations suggest that fusomes are required for the proper formation and asymmetric orientation of mitotic spindles. Moreover, they reinforce the notion that fusomes are required for the four synchronous divisions of the cystoblast leading to cyst formation. In htsl cysts that lack fusomes and fail to incorporate an hts gene product(s) into ring canals following cyst formation, polarized microtubule networks do not form, the dynamics of cytoplasmic dynein are disrupted, and Oskar and Orb RNAs fail to be transported to the future oocyte. These observations support the proposed role of fusomes and ring canals in organizing a polarized microtubule-based transport system for RNA localization that leads to oocyte differentiation (Deng, 1997).

The Drosophila oocyte develops within a cyst of 16 germline cells interconnected by ring canals. One of the 16 germline cells becomes the oocyte, while the other 15 become nurse cells. Polarized, microtubule-based transport of unknown determinants from nurse cells to oocyte is required for oocyte formation. Whether polarity is established during or after cyst formation has not been clear. By following the development of ring canals and the growth of the fusome (a Spectrin containing vesiculated cytoplasmic organelle that interconnects nurse cells and the oocyte), it was possible to analyze how polarity develops in stem cells and dividing cysts. Fusomes were marked with antiserum to adducin-like Hu-li tai shao (Hts). The ring canal was identified by labeling ovaries with antibodies that recognize the actin-binding protein, Anillin (Field, 1995). In Drosophila embryos and tissue culture cells, Anillin is expressed in actively dividing cells and is localized in a cell-cycle-dependent manner. During interphase, it accumulates in the nucleus, but in mitosis, following nuclear envelope breakdown, it is released into the cytoplasm and moves out to the cortex. At telophase, Anillin becomes highly enriched in the cleavage furrow, where it remains until the connection between the sister cells is severed. It is usually not detected in cells that have left the cell cycle. In germline stem cells and cystocytes, Anillin follows the same cell-cycle-dependent pattern of localization; however, it persists in the ring canals that link the cystocytes long after the cells have stopped dividing, at least until the cyst has left the germarium. Anillin also localizes to a transient ring canal that arises when a stem cell divides to produce a daughter stem cell and a cystoblast. When it is first formed, this ring canal is similar in size and shape to the ring canals that link dividing cystocytes. It is not a permanent structure, however; before either cell divides again, the ring canal shrinks in diameter and the hole in its center disappears, severing the connection between the two cells. Usually no detectable Anillin remains at the site of the severed connection, though occasionally a small spot can be found on the membrane of a dividing cystoblast. This stem cell ring canal was identified previously in electron micrographs of Drosophila germaria, but Anillin is its first molecular component to be identified (de Cuevas, 1998).

Having a marker for the stem cell ring canal has allowed the identification of stem cells that have recently completed mitosis, whose fusomes are likely to be in the process of segregating between the two daughter cells. To analyze the behavior of the fusome in these cells, ovaries were fixed and triple-stained with anti-Anillin antibodies, anti-Hts antibodies (which label the fusome) and the DNA dye DAPI; the cells were then examined by immunofluorescence and confocal microscopy. Stem cells were identified by their location in the germarium, directly abutting the base of the terminal filament. The cell cycle phase of a stem cell was identified from its nuclear morphology, from the location of Anillin within the cell, and from the presence or absence of a ring canal. In late interphase, when the stem cell has a high level of Anillin in its nucleus and there is no ring canal attaching it to any other cell, the fusome is spherical in shape and is located at the anterior tip of the stem cell, adjacent to the base of the terminal filament. Throughout mitosis, when Anillin is enriched at the cell cortex, the fusome stays in this location and usually remains spherical, although occasionally it flattens out along the membrane of the stem cell, forming a wedge-shaped structure. After mitosis, when the nuclear membrane has reformed and the cleavage furrow is well advanced, the fusome begins to migrate posteriorly toward the cleavage furrow. At the same time, a small amount of fusomal material accumulates in the nascent ring canal, forming a ‘plug’ in the ring. Although its origin(s) could not be determined from this experiment, it is suggested that the plug is formed at least in part from newly synthesized material, since the original spherical fusome does not become noticeably smaller as it forms. As interphase progresses, the plug enlarges and the original fusome elongates along the anterior-posterior axis of the stem cell, eventually contacting the growing plug. The resulting single fusome then reorganizes and/or accumulates new material until it forms a thick bar-shaped structure that extends through the ring canal. Later, as the ring canal closes, it appears to squeeze the bar-shaped fusome into two pieces. Because of the appearance of the fusome, this has been called the ‘exclamation point’ stage. At this stage, the fusome is usually distributed unequally between the two cells; thus about one-third of it ends up in the cystoblast, and the remainder in the stem cell (de Cuevas, 1998). The bar-shaped fusome and its unequal distribution between the two cells were previously reported by Deng (1997).

Stable intercellular bridges called ring canals form following incomplete cytokinesis, and interconnect mitotically or meiotically related germ cells during spermatogenesis. Ring canals in Drosophila melanogaster males are surprisingly different from those previously described in females. Mature ring canal walls in males lack actin and appear to derive directly from structural proteins associated with the contractile ring. Ring canal assembly in males, as in females, initiates during cytokinesis with the appearance of a ring of phosphotyrosine epitopes at the site of the contractile ring. Following constriction, actin and myosin II disappear. However, at least four proteins present at the contractile ring remain: the three septins (Pnut, Sep1 and Sep2) and anillin. In sharp contrast, in ovarian ring canals, septins have not been detected, anillin is lost from mature ring canals and filamentous actin is a major component. In both males and females, a highly branched vesicular structure, termed the fusome, interconnects developing germ cells via the ring canals and is thought to coordinate mitotic germ cell divisions. In males, unlike females, the fusome persists and enlarges following cessation of the mitotic divisions, developing additional branches during meiosis. During differentiation, the fusome and its associated ring canals localize to the distal tip of the elongating spermatids (Hime, 1996).

The cytoskeletal apparatus of the vertebrate intestinal brush border (BB) has served as a model system for the actin-based cytoskeleton of nonmuscle cells. The structural organization and molecular architecture of the BB cytoskeleton of lepidopteran larvae (Manduca sexta) have been studied. Electron microscopy of the midgut of the 5th instar larvae reveals enterocytes with an apical BB surface comparable to that in the vertebrate intestine, with both microvillar (MV) and terminal web (TW) domains, the latter defined by a zone of organelle exclusion directly beneath the MV. The MV domains contain a bundle of actin filaments, and heavy meromyosin decoration. Two-dimensional gel analysis reveals the presence of multiple isoelectric variants of actin with the major isoform corresponding to the non-muscle actin isoform II, expressed in Drosophila. Like the vertebrate BB, the Manduca BB can be isolated intact from enterocytes by mechanical shear. The MV core proteins: BB myosin I, villin and fimbrin, are present as well as the TW components: spectrin, myosin II and tropomyosin. Immunocytochemical localization of a subset of these proteins at the light microscopic (spectrin) and electron microscopic (actin, villin, spectrin, myosin II, and tropomyosin) level reveals that the molecular architecture of the Manduca BB cytoskeleton is homologous to that found in vertebrates (Bonfanti, 1992).

continued: see Developmental Biology - Adult part 2/2

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