Cytoskeleton

The cytoskelton is a meshwork of protein polymer filaments found in all eukaryotes (reviewed in Botstein et al., 1997). In Saccharomyces, as in other cell types, it serves to organize the cytoplasm, to provide mechanisms for movement of structures such as vesicles or the nucleus during mating, and to maintain cell shape. Three types of filament consisting of distinct proteins are present. Microfilaments (5-7 nm in diameter) are long flexible polymers of actin hundreds of monomers long. Microtubules (25 nm in diameter) consist of tubulin subunits polymerized to form hollow tubes plus other associated proteins. A number of intermediate filaments (about 10 nm in diameter) consisting of fibrous proteins called septins are found at the bud neck. As discussed above, these neck filaments function as a cytokinesis tag or location signal.

Nuclear envelope

Nuclear envelope

Figure 3.8 Representation of the Saccharomyces SPB. The three plate-like plaques of the SPB are shown in cross-section. The central plaque is imbedded in and spans the nuclear envelope. The outer plaque is on the cytoplasmic side of the nuclear envelope and is the attachment site of the cytoplasmic microtubules. It is also the site at which spore wall formation is initiated. The inner plaque lies within the nucleus, and the intranuclear microtubules, those that compose the spindle in a dividing cell, are attached here. A few of the known protein components and their location in this complex structure are indicated. Taken from Botstein et al. (1997). Reproduced with permission from Cold Spring Harbor Laboratory Press

Figure 3.8 Representation of the Saccharomyces SPB. The three plate-like plaques of the SPB are shown in cross-section. The central plaque is imbedded in and spans the nuclear envelope. The outer plaque is on the cytoplasmic side of the nuclear envelope and is the attachment site of the cytoplasmic microtubules. It is also the site at which spore wall formation is initiated. The inner plaque lies within the nucleus, and the intranuclear microtubules, those that compose the spindle in a dividing cell, are attached here. A few of the known protein components and their location in this complex structure are indicated. Taken from Botstein et al. (1997). Reproduced with permission from Cold Spring Harbor Laboratory Press

ACTIN CYTOSKELETON

Saccharomyces actin is an approximately 42 kD protein encoded by ACT] and exhibits high sequence homology to actin from other eukaryotes. It is a globular protein found as a monomer and in polymerized form, i.e. microfilaments. In Saccharomyces polymerized actin is localized to several so-called cortical patches and to long fibers sometimes referred to as actin cables. The cortical patches are associated with the small invaginations of the plasma membrane and the actin cables often appear to extend from these cortical patches and orient parallel to the long length of the cell. The distribution of cortical actin patches changes during the cell cycle and this is beautifully illustrated in Figure 3.9. Similarly, changes in the distribution of cortical actin patches are seen prior to schmoo formation (Marsh & Rose, 1997). These changes in the distribution of cortical actin patches strongly

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Figure 3.9 Confocal imaging of the actin cytoskeleton in budding Saccharomyces. Cells from a dividing population at m vs

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Figure 3.9 Confocal imaging of the actin cytoskeleton in budding Saccharomyces. Cells from a dividing population at m different stages of the cell cycle are shown stained with rhodamine-conjugated phalloidin, which binds to polymerized m actin cortical patches (bright spots) and actin cables (thread-like structures). Localization to the site of bud formation, >

to the growing bud, and to the bud neck during cell separation is clearly evident. Taken from Botstein et al. (1997). Reproduced with permission from Cold Spring Harbor Laboratory Press I

suggest an association with polarized cell growth that includes remodeling of the cell wall, activation of chitin synthase, and microtubule localization.

MICROTUBULE CYTOSKELETON

Microtubules are hollow tubes composed of two highly homologous proteins called a-tubulin (50 kD and encoded by TUB! and TUB3) and /3-tubulin (51 kD and encoded by TUB2). Two classes of microtubules are found in yeast, namely cytoplasmic microtubules and intranuclear microtubules. Both classes are attached to the spindle pole body and extend either into the cytoplasm or the nucleoplasm.

Microtubules are used for the movement of subcellular organelles, most particularly the nucleus. Motor proteins that attach to and move along the microtubule use the microtubule as a tracklike substrate. Two classes of motor proteins have been identified in Saccharomyces, i.e. dyneins and kinesins. Dynein is a large multisubunit protein complex involved in nuclear movement during cell division and mating. The kinesins are smaller protein complexes consisting of a heavy chain with motor activity and a light chain. At least five genes encoding kinesin components have been identified in Saccharomyces. Motor proteins can bridge between two microtubules and move these past one another thereby creating movement potentially in either direction depending on the motor proteins involved. Dynein moves toward the SPB direction (negative direction) whereas different kinesins are capable of moving in either the plus or minus direction. It is also likely that some lengthening of the microtubules themselves occurs during spindle elongations in anaphase.

MICROTUBULE MORPHOLOGY IN CELL DIVISION AND MATING

In the unbudded cell, several microtubules can be seen to extend from the SPB into the nucleus and in various directions into the cytoplasm. During budding, both cytoplasmic and intranuclear microtubules undergo dramatic changes beginning with the formation of a second SPB in early G1 that is complete by the start of S. The array of intranuclear microtubules extending from the two SPBs is collectively called the spindle. Detailed structural analysis of the Saccharomyces spindle during mitosis has been carried out by Mark Winey and coworkers and is reviewed in Winey & O'Toole (2001). During the S phase the two SPBs slowly move apart and by G2 are located at opposite poles of the nucleus. Short intranuclear microtubules attach to the inner plaque of the SPB at one end and to the chromosomal kineto-chore. Other longer intranuclear microtubules extend from one SPB to the other. Chromosome separation, i.e. anaphase, is achieved in some degree by the shortening of the kinetochore-bound microtubules but mostly by an increase in the pole-to-pole distance between the two SPBs. This lengthening of the spindle is dependent on microtubule growth processes and motor proteins.

Throughout these intranuclear events of mitosis, the cytoplasmic microtubules are involved in localizing the nucleus to the bud neck and directing the future daughter cell nucleus into the bud. In Figure 3.10 one can see the nuclear oscillations that occur in the mother cell prior to anaphase and the apparent searching process is carried out by the bud-directed cytoplasmic microtubules, perhaps for an

Figure 3.10 Microtubule orientation in dividing and nondividing living cells. The cells shown are expressing a fully functional GFP-tubulin fusion protein allowing microtubules to be visualized in living cells. A time course of changes in microtubule shape and orientation is shown for an unbudded cell (panel B) and for a large-budded cell (panel C). The photographs demonstrate the process of cytoplasmic microtubule searching that ultimately localizes the nucleus to the bud neck. Taken from Carminati and Stearns (1997). Reproduced with permission of the Rockefeller University Press

Figure 3.10 Microtubule orientation in dividing and nondividing living cells. The cells shown are expressing a fully functional GFP-tubulin fusion protein allowing microtubules to be visualized in living cells. A time course of changes in microtubule shape and orientation is shown for an unbudded cell (panel B) and for a large-budded cell (panel C). The photographs demonstrate the process of cytoplasmic microtubule searching that ultimately localizes the nucleus to the bud neck. Taken from Carminati and Stearns (1997). Reproduced with permission of the Rockefeller University Press attachment site in the bud. These microtubules determine the orientation of the spindle and ensure that spindle lengthening occurs through the bud neck (Rong, 2000). As a result of spindle lengthening the nucleus acquires an elongated hourglass shape that extends through the bud neck and places one end in the mother cell and the other in the bud. Separation into two nuclei, the two-budded stage, marks the end of the division phase of the cell cycle and the beginning of G1 of the next cell cycle.

Various cytological events are used as mileposts to assess progress through the cell cycle. These events include SPB duplication, localization of cortical actin patches, initiation of bud growth, size of bud, localization of the nucleus to the bud neck, nuclear shape or spindle length and shape, and number of nuclei. These events have been carefully correlated with the stages of the cell cycle and thus can be used to determine the timing of another process, such as the requirement for a particular gene function, in the cell cycle without the need to measure DNA levels or other more cumbersome assays.

Rose (1996) reviews the role of microtubules in nuclear fusion during mating. Figure 3.7 shows this process. The SPB of each parental nucleus orients toward the growing schmoo projection and, as the cytoplasms of the two mating cells fuse, the cytoplasmic microtubules attached to the two SPBs intertwine. Motor proteins associated with the cytoplasmic microtubules move the two nuclei together and allow fusion to occur. Following fusion, the cytoplasmic microtubules of the newly formed diploid nucleus direct it into the new bud forming at the junction of the parental cells.

PLASMA MEMBRANE, ENDOPLASMIC RETICULUM, GOLGI COMPLEX, VACUOLE, AND MEMBRANE TRAFFICKING

The endoplasmic reticulum (ER), the Golgi complex, the plasma membrane, and the vacuole must be considered together. These physically separated subcellular compartments are intimately interconnected as a result of the movement of membrane that occurs from one compartment to the next via membrane-bounded vesicles carrying a cargo of proteins and other macromolecules such as cell wall components. This movement of membrane and proteins is sometimes referred to as membrane trafficking. The secretory pathway flows from the ER to the Golgi complex where it branches, sending vesicles either to the plasma membrane (exocytosis) or to the vacuole. It is estimated that from 10% to 20% of a putative 6000 Saccharomyces proteins are either residents or passengers of the secretory pathway (Kaiser et al., 1997). Membrane and selected proteins are also removed from the plasma membrane and directed to the vacuole via a process referred to as endocytosis. For an in-depth discussion of the structure, function, and biosynthesis of these compartments and pathways the reader is directed to reviews by Kaiser et al. (1997) and Jones et al. (1997). Figure 3.11 is a transmission EM image of Saccharomyces showing the various organelles involved in the secretory pathway.

ENDOPLASMIC RETICULUM

The endoplasmic reticulum is the site of protein synthesis of secreted and integral membrane proteins localized to the various compartments of the secretory pathway. The ER is contiguous with the outer membrane of the nuclear envelope where it is studded with ribosomes involved in the synthesis and translocation of secreted proteins across the ER membrane into the lumen of the ER or the insertion of newly synthesized integral membrane proteins into the ER membrane. Extensions of the ER can be seen extending in from the outer nuclear membrane into the cytoplasm out as far as the edges of the cytoplasm and these can be visualized as a discontinuous

Figure 3.11 The organelles of the secretory pathway. A transmission electron micrograph of a cell with a newly forming bud illustrates the various subcellular organelles of the secretory pathway and their organization within the cytoplasm. The ER can be seen to extend from the outer membrane of the nuclear envelope (Nuc indicates the nucleus) in projections toward the surface of the cell. The ER also underlies the plasma membrane. Plasma membrane and cell wall growth is concentrated in the bud, which is packed with secretory vesicles (sv). The Golgi is localized to the bud neck region of the mother cell. Saccharomyces has only a single large vacuole (Vac) but this appears to be doubled in this micrograph because of a sectioning artifact. Taken from Kaiser et al. (1997). Reproduced by permission of Cold Spring Harbor Laboratory Press

Figure 3.11 The organelles of the secretory pathway. A transmission electron micrograph of a cell with a newly forming bud illustrates the various subcellular organelles of the secretory pathway and their organization within the cytoplasm. The ER can be seen to extend from the outer membrane of the nuclear envelope (Nuc indicates the nucleus) in projections toward the surface of the cell. The ER also underlies the plasma membrane. Plasma membrane and cell wall growth is concentrated in the bud, which is packed with secretory vesicles (sv). The Golgi is localized to the bud neck region of the mother cell. Saccharomyces has only a single large vacuole (Vac) but this appears to be doubled in this micrograph because of a sectioning artifact. Taken from Kaiser et al. (1997). Reproduced by permission of Cold Spring Harbor Laboratory Press cisterna underlying the plasma membrane (see Figures 3.11 and 3.12). Fluorescence microscropy of cells expressing a GFP fusion to the ER-localized protein Gsf2p illustrates the cellular position of the Saccharomyces ER (Figure 3.12).

Various protein modifications occur in the ER. Cleavage of the signal peptide occurs during protein translocation. The addition of N-linked and O-linked carbohydrate groups is initiated in the ER as well as the addition of GPI anchors. Protein folding and disulfide bond formation occur here. This is particularly important for certain membrane proteins and for the formation of multiprotein complexes in the ER lumen. Improperly folded integral membrane proteins are targets of proteolysis via quite specific pathways (Kaufman, 1999) and the ER is a site of regulated protein degradation.

Figure 3.12 GFP-fluorescence imaging of the endoplasmic reticulum. The cells shown are expressing a GFP fusion to the ER-localized protein Gsf2p. The subcellular location of the fully active GFP-Gsf2p fusion is visualized by confocal image analysis. Fluorescence is observed in the region closely surrounding the nucleus, in cytoplasmic threads extending from the nucleus, and in the region underlying the plasma membrane. Taken from Sherwood & Carlson (1999). Reproduced by permission of the National Academy of Sciences, USA

Figure 3.12 GFP-fluorescence imaging of the endoplasmic reticulum. The cells shown are expressing a GFP fusion to the ER-localized protein Gsf2p. The subcellular location of the fully active GFP-Gsf2p fusion is visualized by confocal image analysis. Fluorescence is observed in the region closely surrounding the nucleus, in cytoplasmic threads extending from the nucleus, and in the region underlying the plasma membrane. Taken from Sherwood & Carlson (1999). Reproduced by permission of the National Academy of Sciences, USA

GOLGI COMPLEX

The Golgi complex in Saccharomyces is a grouping of several membrane-bound cisternae. Despite the fact that the different cisternae cannot be distinguished microscopically, functional analysis clearly indicates that the Saccharomyces Golgi consists of three compartments referred to as the cis-, medial-, and trans-Golgi. Proteins enter the c/.v-Golgi from the ER and proceed to the medial- and trans-Golgi where they receive sequential modifications, including the addition of outer-chain mannose residues. Mature secretory proteins are selectively packaged in the trans-Golgi into vesicles and proceed from here to either the plasma membrane or the vacuole (with an intermediate stop in a prevacuolar compartment called the endosome).

VACUOLE

The Saccharomyces vacuole is a large centrally located single membrane-bound organelle easily visualized by Nomarski optics and is the equivalent of the mammalian lysosome (see Figure 2.5). Jones et al. (1997) provides an excellent overview of vacuolar function and biosynthesis. The vacuole contains a variety of proteases and hydrolases for the degradation of polysaccharides and RNA. It serves as a storage site for ions such as Ca2+ and amino acids and the vacuolar membrane contains pumps for these ions/molecules as well as a proton pump for regulating vacuolar pH. The vacuole plays a very important role in the physiological transition from one growth state to another such as occurs during changes in nutritional conditions or entry into the stationary phase.

Proteins get to the vacuole by a number of different pathways. Already mentioned is the movement of protein cargo-containing vesicles from the Golgi. Some of

Figure 3.13 Overview of exocytosis and endocytosis. The movement of membrane through the secretory pathway and from the membrane to the vacuole via endocytosis is shown. N, nucleus; ER, endoplasmic reticulum: G, Golgi complex; LE, late endosome (prevacuolar compartment); EV, endocytic vesicle; SV, secretory vesicle; I, invagination of the plasma membrane (clathrin-coated pit); V, vacuole. Note that the forward and retrograde movement within the Golgi complex (cis-, medial-, and trans-Golgi compartments) as well as retrograde movement from the Golgi to the ER described in the text also involves very specialized vesicles, but these are not illustrated in the diagram for simplicity. Taken from Walker (1998). Reproduced with permission from John Wiley & Sons, Limited

Figure 3.13 Overview of exocytosis and endocytosis. The movement of membrane through the secretory pathway and from the membrane to the vacuole via endocytosis is shown. N, nucleus; ER, endoplasmic reticulum: G, Golgi complex; LE, late endosome (prevacuolar compartment); EV, endocytic vesicle; SV, secretory vesicle; I, invagination of the plasma membrane (clathrin-coated pit); V, vacuole. Note that the forward and retrograde movement within the Golgi complex (cis-, medial-, and trans-Golgi compartments) as well as retrograde movement from the Golgi to the ER described in the text also involves very specialized vesicles, but these are not illustrated in the diagram for simplicity. Taken from Walker (1998). Reproduced with permission from John Wiley & Sons, Limited these vesicles contain vacuolar resident proteins on their way to their final destination. More recently it appears that some plasma membrane proteins may be redirected to the vacuole for degradation in response to physiological changes (Roberg et al., 1997). Vesicles formed at the plasma membrane by endocytosis are targeted to the vacuole for degradation by the vacuolar enzymes. Both vesicles from the Golgi and those from the plasma membrane do not go directly to the vacuole but go first to a prevacuolar compartment called the endosome. The endosome may go through a maturation process before finally fusing with the vacuolar membrane and delivering its contents to the vacuole for degradation. Autophagy is another route to the vacuole. Cytoplasmic proteins and even organelles are surrounded by membrane and these autophagosomes are taken into the vacuole where they are degraded and recycled. Finally, a so-called cytoplasm-to-vacuole targeting pathway, or cvt pathway, which at least partially overlaps with the autophagy pathway has been described (Lang et al., 2000).

MEMBRANE TRAFFICKING

An overview of the secretory pathway and endocytosis is shown in Figure 3.13 and reviewed in Kaiser et al. (1997). This figure illustrates only forward movement through the secretory pathway but it should be noted that retrograde movement of vesicles also occurs from the Golgi to the ER as well as from the late Golgi to the cz.s-Golgi. The purpose of this retrograde movement is to return proteins that were carried along with the cargo from one compartment to the next back to their home compartment for reutilization.

It is most important to keep in mind that movement from compartment to compartment, whether forward or retrograde, is carried out by specialized vesicles. Vesicles are classified based on the protein 'coat' found on the cytoplasmic side of the vesicle. Vesicles directed to the vacuole, i.e. endocytic vesicles and vesicles formed by the trans-Golgi, are clathrin-coated vesicles. COPI vesicles that are coated by a protein complex called coatomer are involved in intra-Golgi transport and retrograde transport from the Golgi to the ER. Vesicles from the ER to the Golgi are coated with a set of proteins called COPII and are referred to as COPII vesicles.

The specificity of the interaction between the vesicle and the target compartment is controlled by large protein complexes called SNARE complexes located within the membranes of the vesicle, called the v-SNARE, and the target membrane, called the t-SNARE. These can be thought of as a lock and key mechanism to ensure that a particular vesicle carrying a cargo intended for a specific compartment fuses with that compartment and only that compartment (reviewed in Kaiser et ah, 1997).

MITOCHONDRION

As in other eukaryotes, the Saccharomyces mitochondrion is a double membrane organelle. Enzymes of lipid metabolism are located in the outer membrane. The inner membrane, called the cristae, is highly convoluted and contains the enzymes involved in respiration and ATP synthesis. The enzymes of the citric acid cycle and of fatty acid oxidation are found in the matrix along with the mitochondrial DNA and the mitochondrial protein synthetic machinery. Biosynthesis of the mitochondrion involves both nuclearly encoded proteins and RNAs and proteins encoded by the mtDNA.

Under different growth conditions, such as growth on fermentable versus non-fermentable carbon source or the presence or absence of oxygen, the number of Saccharomyces mitochondria per cell will vary as well as the number of copies of the mtDNA. Figure 3.14 shows a three-dimensional reconstruction of the mitochondria produced by confocal analysis of Saccharomyces cells grown in different carbon sources. Saccharomyces is a facultative anaerobe and when grown on fermentable carbon sources like glucose does not require an active mitochondrion for ATP production. Under these conditions, the extent of the crista development is greatly reduced and the mitochondria are sometimes referred to as ghosts.

PEROXISOME

Peroxisomes are single-membrane organelles that contain at least one enzyme for the production of hydrogen peroxide, for example catalase, and enzymes to catalyze its decomposition. The reader is directed to Lazarow & Kunau (1997) for detailed information on the structure, function, and biosynthesis of peroxisomes. The expression of genes encoding peroxisomal proteins is repressed by growth on glucose. Therefore the number of peroxisomes is usually quite low in glucose-grown cells. A major function of peroxisomes is the /^-oxidation of fatty acids and peroxisomes are best visualized in cells grown in oleic acid or other fatty acids that induce the expression of peroxisomal proteins.

Figure 3.14 Three-dimensional images of mitochondria. Scanning confocal microscropy was used to create the three-dimensional images of the shape and distribution of mitochondria in Saccharomyces. The cell shown in panel (a) was grown in ethanol, a nonfermentable carbon source requiring active mitochondria for utilization. The cell in panel (b) was grown in a high concentration of glucose. Under this condition Saccharomyces ferments the sugar producing ethanol, carbon dioxide, and ATP, and does not require fully elaborated mitochondria. Taken from Visser et al. (1995). Reproduced with kind permission from Kluwer Academic Publishers

Figure 3.14 Three-dimensional images of mitochondria. Scanning confocal microscropy was used to create the three-dimensional images of the shape and distribution of mitochondria in Saccharomyces. The cell shown in panel (a) was grown in ethanol, a nonfermentable carbon source requiring active mitochondria for utilization. The cell in panel (b) was grown in a high concentration of glucose. Under this condition Saccharomyces ferments the sugar producing ethanol, carbon dioxide, and ATP, and does not require fully elaborated mitochondria. Taken from Visser et al. (1995). Reproduced with kind permission from Kluwer Academic Publishers

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Broach, J., J. Pringle, & E. Jones, editors (1997) The Molecular and Cellular Biology of the Yeast Saccharomyces. Vol. 3: Cell Cycle and Cell Biology. Cold Spring Harbor Press, New York.

Carminati, J.L. & T. Stearns (1997) Microtubules orient the mitotic spindle in yeast through dynein-dependent interactions with the cell cortex. J. Cell Biol. 138: 629-641.

Gimeno, C.J., P.O. Ljungdahl, C.A. Styles, & G.R. Fink (1992) Unipolar cell divisions in the yeast S. cerevisiae lead to filamentous growth: regulation by starvation and RAS. Cell 68: 1077-1090.

Griffin, D.H. (1994) Fungal Physiology, 2nd edition. Wiley-Liss Inc., New York.

Johnson, D.I. (1999) Cdc42: An essential Rho-type GTPase controlling eukaryotic cell polarity. Microbiol. Mol. Biol. Rev. 63: 54-105.

Jones, E.B., G.C. Webb, & M.A. Hiller (1997) Biogenesis and function of the yeast vacuole. In The Molecular and Cellular Biology of the Yeast Saccharomyces. Vol. 3: Cell Cycle and Cell Biology, Broach, J., J. Pringle, & E. Jones, editors. Cold Spring Harbor Press, New York, pp. 363-470.

Kaufman, R.J. (1999) Stress signalling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls. Genes Dev. 13: 1211-1233.

Kaiser, C.A., R.E. Gimeno, & D.A. Shaywitz (1997) Protein secretion, membrane biogenesis, and endocytosis. In The Molecular and Cellular Biology of the Yeast Saccharomyces. Vol. 3: Cell Cycle and Cell Biology, Broach, J., J. Pringle, & E. Jones, editors. Cold Spring Harbor Press, New York, pp. 91-227.

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Lang, T., S. Reiche, M. Straub, M. Bredschneider, & M. Thumm (2000) Autophagy and the cvt pathway both depend on AUT9. J. Bacteriol. 182: 2125-2133.

Lazarow, P.B. & W. Kunau (1997) Peroxisomes. In The Molecular and Cellular Biology of the Yeast Saccharomyces. Vol. 3: Cell Cycle and Cell Biology, Broach, J., J. Pringle, & E. Jones, editors. Cold Spring Harbor Press, New York, pp. 547-606.

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Genetic Techniques for Biological Research Corinne A. Michels Copyright © 2002 John Wiley & Sons, Ltd ISBNs: 0-471-89921-6 (Hardback); 0-470-84662-3 (Electronic)

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