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Abstract. Bud emergence, spindle pole body duplication and DNA replication are all dependent on the activation of the CDC28 protein kinase at the Start point in the G1 phase of the cell cycle. Bud emergence requires polarization of the cytoskeleton and secretory vesicles to a specific site on the cell surface. Cdc28p activated by Gl-cyclins triggers polarization of actin to the site of bud emergence and favors apical bud growth (Lew, D. J., and S. I. Reed. 1993. J. Cell Biol. 120:1305-1320 C ELL reproduction involves cell cycle-specific events occurring both in the nucleus and in the cytoplasm. In the budding cell cycle of Saccharomyces cerevisiae, a number of stage-specific events direct the position and the morphology of the bud. These events include the choice of a bud site, the localization of a specific group of proteins to the site from which the bud will emerge, and the polarization of cell surface growth to the new bud site. Assembly of the bud-site complex is required for the polarization of the yeast cytoskeleton and the targeting of secretory vesicles to the bud site (for reviews see references 9, 12, 32).The initial growth of the bud is highly polarized. Actin cortical patches (22), calmodulin (6), and the SPA2 protein (53, 54) are localized to the tips of the bud during this period of apical growth. Actin localization and bud growth are less polarized later in the cell cycle. The timing of this shift and the degree of delocalization determine bud shape, with isotropic growth favoring spherical forms (28,55).The entry into a new cell cycle involves the coordinated but independent pathways of bud initiation, spindle pole body duplication, and nuclear DNA synthesis (43). The triggering of these pathways all require the activation of the CDC28 protein kinase in the G1 phase of the ceil cycle (45). This regulatory point of the cell cycle has been baptized "Start" (18). The manner in which the CDC28 ldnase promotes bud formation at Start is not well defined. Lew and Reed (28) have shown that actin polarization can be triggered by the kinase activity of Cdc28p complexed with G1 cyclins, and they mapped the time at which actin polarization occurs in their synchronized cells to be soon after Start. Localization of Cdc3p (23) and Spa2p (54) to the presumptive bud site occurs substantially before bud emergence, but the precise timing of these events relative to CDC28 kinase activation at Start was not examined. However, cdc28 mutants at their restrictive temperature and wild-type cells treated with mating pheromone can polarize their growth to form a projection in the apparent absence of CDC28 kinase activity (28,31,54). These observations suggest that a pathway for polarizing growth independent of Cdc28p also exists in S. cerevisiae.We isolated a mutation of the SLT2 (MPKI) MAP kinase gene that augments the division defect of cells expressing a partially inactivated cdc28 mutant. We show that sit2 mutants exhibit defects in cell polarity and accumulate secretory vesicles. We suggest that Slt2p acts downst...

Glutaraldehyde-fixed testes were stained "en bloc" with the Ur-Pb-Cu technique of Thiéry and Rambourg ('76) or post-fixed and stained with the osmium tetroxide-potassium ferrocyanide method of Karnovsky ('71). Thin or thick (up to 3 micron) sections were examined with the Philips (301 or 400) EM or the high voltage EM. Stereopairs were prepared with photographs of tilted specimens (+/- 7 degrees). At low magnification, in thick sections (0.5-3 micron) stained with Ur-Pb-Cu, the whole Golgi apparatus formed a single network of interconnected wavy ribbon or platelike structures extending from the juxtanuclear region toward the apex of the cell. At higher magnifications, with the two staining techniques, this Golgi network showed two distinct types of regions: the "saccular region" corresponding to the conventional stack of saccules and the "intersaccular connecting region" made up of anastomotic tubules which bridge adjacent stacks. In the saccurlar regions, there was, on the cis-face of the stack, a tight polygonal meshwork of anastomotic tubules (osmiophilic element). Underlying it there were three to seven closely apposed saccules perforated with pores of various diameters, and finally, on the trans-face, a network of tubules was usually connected to the last saccule of the stack, which seemed to peel off" from the pile. The intersaccular connecting regions showed proximal and distal zones with regard to the associated stacks. The proximal zone was made up of superimposed and parallel polygonal networks of membranous tubules which were continuous with corresponding saccules of the stack. In the distal zone they interdigitated, intertwined, anastomosed and bridged adjacent saccular regions; others turned at right angles and established connections with tubular extensions arising at various levels of the same stack. While cisternae of endoplasmic reticulum were contiguous with tubules or saccules located on the transface of the Golgi apparatus, a close association between the ER cisternae and the cis-face of the stacks was not usually observed.

Periodic acid-silver methenamine, a fairly specific technique for glycoprotein detection, was used to stain a variety of rat tissues, in the hope of confirming the existence of a carbohydrate-rich "cell coat" at the surface of mammalian cells. It was found that nearly all cells are coated by a thin layer of stained material. Around fibrocytes and migrating blood cells, the layer is uniform and merges with the ground substance. In the nervous system, cells and processes are surrounded with a layer whose density increases in synaptic clefts. Around epithelial cells, the layer outlines apical microvilli, follows lateral interspaces, and extends between cells and basement membrane. The layer is continuous with the middle plate of desmosomes and can be followed within the wide portion of terminal bars. In contrast, staining usually vanishes when two adjacent plasma membranes fuse to form tight junctions. These findings indicate that the stained layer is a "cell coat" located outside the plasma membrane. Since the cell coat is also stained by colloidal thorium, a technique for detection of acidic carbohydrates, this structure presumably contains not only glycoprotein(s) but also acidic residues. The carbohydrates may play a role in holding cells together and in controlling the interactions between cells and environment.

Two types of chloride cells were identified in the gill epithelium of freshwater-adapted guppies. One type, referred to as an "alpha-chloride cell," was a pale, elongated cell located at the base of the secondary lamella in close contact with the arterioarterial pillar capillaries. In its cytoplasm, membranous tubules in continuity with its basolateral plasma membrane formed an extended tridimensional network. The vesiculotubular system (Pisam: Anat. Rec. 200:401-414, 1981) consisted of a few tubules and vesicles located next to the apical plasma membrane. A second type, referred to as a "beta-chloride cell," was a darker, ovoid cell located in the interlamellar region of the primary epithelium facing the central venous sinus. Membranous tubules in continuity with the basolateral plasma membrane were unevenly distributed in the cytoplasm. A prominent vesiculotubular system composed of numerous vesicles and tubules was found between the Golgi apparatus and the apical surface. During seawater adaptation, the alpha-chloride cells increased in size and progressively transformed into characteristic "seawater alpha-chloride cells" with a well-developed, regular, tight tubular network and numerous vesicles and tubules of the vesiculotubular system accumulated below the apical pit. The beta-chloride cells underwent a progressive degeneration and disappeared. Thus, in freshwater-adapted guppies, there are two types of chloride cells, alpha and beta, respectively, related to the arterial and the venous vessels, whereas in seawater-adapted fishes, a single type of cell, the alpha-chloride cell, was related to both the arterial and venous channels.

Sections of glutaraldehyde-fixed lumbar dorsal root ganglia of the rat were examined in the electron microscope following impregnation with the uranyl-lead-copper technique or postfixation in potassium ferrocyanide-reduced osmium. Three types of ganglion cells (A, B, C) were identified on the basis of their size and the distribution of their organelles. They were further subdivided into six subtypes according to the arrangement and three-dimensional organization of the Nissl bodies and Golgi apparatus in the perikarya. Type A1 cells were large, clear neurons in which Nissl bodies, separated from each other by pale narrow strands of cytoplasm containing small stacks of Golgi saccules and rod-like mitochondria, were evenly distributed throughout the perikaryon. In type A2, the Nissl bodies assumed a similar distribution but were separated by much wider strands of cytoplasm. Type A3, the smallest of the type A category, displayed densely packed Nissl bodies and long stacks of Golgi saccules which formed a perinuclear ring in the midportion of the perikaryon. Type B cells were smaller and showed a concentric zonation of their organelles. In type B1, large Nissl bodies located in an outer cytoplasmic zone were made of long piles of parallel cisternae interrupted by curved Golgi stacks. Type B2 was characterized by a ring-like Golgi apparatus separating the perikaryon in a cortical zone composed mainly of Nissl substance and a juxtanuclear zone containing mitochondria and smooth endoplasmic reticulum. Type C cells were the smallest of the ganglion cells and contained small, poorly demarcated Nissl bodies and a juxtanuclear Golgi apparatus.

Arf GTPases regulate both the morphological and protein sorting events that are essential for membrane trafficking. Guanine nucleotide exchange factors (GEFs) specific for Arf proteins determine when and where Arf GTPases will be activated in cells. The yeast Gea2p Arf GEF is a member of an evolutionarily conserved family of high molecular mass Arf GEFs that are peripherally associated with membranes. Nothing is known about how these proteins are localized to membranes, and few direct binding partners have been identified. In yeast, Gea2p has been implicated in trafficking through the Golgi apparatus and in maintaining Golgi structure. A major function of the Golgi apparatus is the packaging of cargo into secretory granules or vesicles. This process occurs through a series of membrane transformation events starting with fenestration of a saccular membrane, and subsequent remodeling of the fenestrated membrane into a mesh-like tubular network. Concentration of secretory cargo into nodes of the tubular network leads to enlargement of the nodes, which correspond to forming vesicles/granules, and thinning of the surrounding tubules. The tubules eventually break to release the secretory vesicles/granules into the cytoplasm. This process is highly conserved at the morphological level from yeast to mammalian cells. Drs2p, a multi-span transmembrane domain protein and putative aminophospholipid translocase, is required for the formation of a class of secretory granules/vesicles in yeast. Here we show that Drs2p interacts directly with Gea2p, both in vitro and in vivo. We mapped the domain of interaction of Drs2p to a 20-amino-acid region of the C-terminal cytoplasmic tail of the protein, adjacent to a region essential for Drs2p function. Mutations in Gea2p that abolish interaction with Drs2p are clustered in the C-terminal third of the Sec7 domain, and are important for Gea2p function. We characterize one such mutant that has a thermosensitive phenotype, and show that it has morphological defects along the secretory pathway in the formation of secretory granules/vesicles.

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