Effects of Human Deafness γ-Actin Mutations (DFNA20/26) on Actin Function
2006; Elsevier BV; Volume: 281; Issue: 29 Linguagem: Inglês
10.1074/jbc.m601514200
ISSN1083-351X
AutoresKeith E. Bryan, Kuo‐Kuang Wen, Mei Zhu, Nanna Dahl Rendtorff, Michael D. Feldkamp, Lisbeth Tranebjærg, Karen H. Friderici, Peter A. Rubenstein,
Tópico(s)S100 Proteins and Annexins
ResumoSix point mutations in non-muscle γ-actin at the DFNA20/26 locus cause autosomal dominant nonsyndromic hearing loss. The molecular basis for the hearing loss is unknown. We have engineered each γ-actin mutation into yeast actin to investigate the effects of these mutations on actin function in vivo and in vitro. Cells expressing each of the mutant actins as the sole actin in the cell were viable. Four of the six mutant strains exhibited significant growth deficiencies in complete medium and an inability to grow on glycerol as the sole carbon source, implying a mitochondrial defect(s). These four strains exhibited abnormal mitochondrial morphology, although the mtDNA was retained. All of the mutant cells exhibited an abnormally high percentage of fragmented/non-polarized actin cables or randomly distributed actin patches. Five of the six mutants displayed strain-specific vacuole morphological abnormalities. Two of the purified mutant actins exhibited decreased thermal stability and increased rates of nucleotide exchange, indicative of increased protein flexibility. V370A actin alone polymerized abnormally. It aggregated in low ionic strength buffer and polymerized faster than wild-type actin, probably in part because of enhanced nucleation. Mixtures of wild-type and V370A actins displayed kinetic properties in proportion to the mole fraction of each actin in the mixture. No dominant effect of the mutant actin was observed. Our results suggest that a major factor in the deafness caused by these mutations is an altered ability of the actin filaments to be properly regulated by actin-binding proteins rather than an inability to polymerize. Six point mutations in non-muscle γ-actin at the DFNA20/26 locus cause autosomal dominant nonsyndromic hearing loss. The molecular basis for the hearing loss is unknown. We have engineered each γ-actin mutation into yeast actin to investigate the effects of these mutations on actin function in vivo and in vitro. Cells expressing each of the mutant actins as the sole actin in the cell were viable. Four of the six mutant strains exhibited significant growth deficiencies in complete medium and an inability to grow on glycerol as the sole carbon source, implying a mitochondrial defect(s). These four strains exhibited abnormal mitochondrial morphology, although the mtDNA was retained. All of the mutant cells exhibited an abnormally high percentage of fragmented/non-polarized actin cables or randomly distributed actin patches. Five of the six mutants displayed strain-specific vacuole morphological abnormalities. Two of the purified mutant actins exhibited decreased thermal stability and increased rates of nucleotide exchange, indicative of increased protein flexibility. V370A actin alone polymerized abnormally. It aggregated in low ionic strength buffer and polymerized faster than wild-type actin, probably in part because of enhanced nucleation. Mixtures of wild-type and V370A actins displayed kinetic properties in proportion to the mole fraction of each actin in the mixture. No dominant effect of the mutant actin was observed. Our results suggest that a major factor in the deafness caused by these mutations is an altered ability of the actin filaments to be properly regulated by actin-binding proteins rather than an inability to polymerize. Hearing depends on sound-dependent distortion of specialized mechanosensory hair cells within the cochlea of the inner ear. In these cells, staircase arrangements of 20–300 hair-like receptors called stereocilia protrude from the apical surface. Mechanical deflection of these structures results in the opening of gated ion channels located on the surface of these protrusions. The result is the conversion of sound-dependent distortion into the propagation of neural signals that are relayed to the brain (1.Hudspeth A.J. Nature. 1989; 341: 397-404Crossref PubMed Scopus (632) Google Scholar). Hair cell function is contingent upon proper functioning of the actin cytoskeleton. Each mature stereocilium contains a core of actin filaments closely packed into rigid hexagonal bundles cross-linked by several actin-binding proteins (2.Tilney L.G. Egelman E.H. DeRosier D.J. Saunder J.C. J. Cell Biol. 1983; 96: 822-834Crossref PubMed Scopus (125) Google Scholar). These filaments not only regulate the three-dimensional aspects of the stereocilia such as height and diameter, but also provide the rigidity necessary for proper functioning of the stereocilium. The stereocilia are embedded in a dense gel-like meshwork of actin filaments with random polarity called the cuticular plate (3.DeRosier D.J. Tilney L.G. J. Cell Biol. 1989; 109: 2853-2867Crossref PubMed Scopus (46) Google Scholar). This structure provides a base in which the stereocilia are anchored and assists in maintaining their erect positions. Encircling each hair cell near its apical surface is the zonula adherens, a circumferential belt of actin filaments that runs parallel to the plasma membrane (4.Hirokawa N. Tilney L.G. J. Cell Biol. 1982; 95: 249-261Crossref PubMed Scopus (248) Google Scholar). These rings form focal contacts with neighboring supporting cells and provide tension across the apical hair cell surface, stabilizing the cuticular plate. Structural integrity and proper function of the cytoskeletal complex depend on the interplay between actin filaments and a number of actin-binding proteins to stabilize the stereocilium, to control its length, and to help organize the cuticular plate. Examples of a few of these actin-binding proteins include spectrin, fimbrin, espin, tropomyosin, harmonin, and formin (5.Muller U. 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However, the functions of these myosins in the hair cell are only partially elucidated at this time. The actin isoform distribution in the hair cells is unusual. Both non-muscle β- and γ-actins are found, but unlike the case for most non-muscle cells, the γ-isoform is the predominant isoform by an ∼2:1 margin (17.Furness D.N. Katori Y. Mahendrasingam S. Hackney C.M. Hear. Res. 2005; 207: 22-34Crossref PubMed Scopus (45) Google Scholar, 18.Hofer D. Ness W. Drenckhahn D. J. Cell Sci. 1997; 110: 765-770Crossref PubMed Google Scholar). The primary sequence of these two isoforms differs at only four amino acid positions. The most striking difference between these two actin isoforms is at the N terminus of the protein, where β-actin contains three aspartate residues, and γ-actin contains three glutamate residues (19.Vandekerckhove J. Weber K. Arch. Int. Physiol. Biochim. 1978; 86: 891-892PubMed Google Scholar). The distribution of these isoforms is also nonrandom. γ-Actin is found throughout the hair cell (17.Furness D.N. Katori Y. Mahendrasingam S. Hackney C.M. Hear. Res. 2005; 207: 22-34Crossref PubMed Scopus (45) Google Scholar, 18.Hofer D. Ness W. Drenckhahn D. J. Cell Sci. 1997; 110: 765-770Crossref PubMed Google Scholar). However, there is a divergence of opinion concerning the localization of β-actin. Hofer et al. (18.Hofer D. Ness W. Drenckhahn D. J. Cell Sci. 1997; 110: 765-770Crossref PubMed Google Scholar) argued that β-actin is confined to the stereocilium, whereas Furness et al. (17.Furness D.N. Katori Y. Mahendrasingam S. Hackney C.M. Hear. Res. 2005; 207: 22-34Crossref PubMed Scopus (45) Google Scholar) reported that β-actin is found in both the cuticular plate (∼30%) and the stereocilium (∼15%). It was recently discovered that six point mutations in the non-muscle γ-actin gene cause hearing loss (20.Zhu M. Yang T. Wei S. DeWan A.T. Morell R.J. Elfenbein J.L. Fisher R.A. Leal S.M. Smith R.J. Friderici K.H. Am. J. Hum. Genet. 2003; 73: 1082-1091Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 21.van Wijk E. Krieger E. Kemperman M.H. De Leenheer E.M.R. Huygen P.L.M. Cremers C.W.R.J. Cremers F.P.M. Kremer H. J. Med. Genet. 2003; 40: 879-884Crossref PubMed Scopus (98) Google Scholar, 22.Rendtorff, N. D., Zhu, M., Fagerheim, T., Antal, T. L., Jones, M.-P., Teslovish, T. M., Gillanders, E. M., Barmada, M., Teig, E., Trent, J. M., Friderici, K. H., Stephan, D. A., and Tranebjaerg, L. (2006) Eur. J. Hum. Genet., in pressGoogle Scholar). These mutations are autosomal dominant; one normal and one mutant γ-actin gene are expressed inside the cell. These mutations are T89I, K118M, P264L, T278I, P332A, and V370A, and in each case, the chemical nature of the mutated residue is very different from that of the residue originally present. All are located in subdomains 1 and 3 of actin, as shown in Fig. 1. Two of these (P332A and P264L) fall near the adenine-binding site on the actin and might affect actin monomer dynamics. Four of these (K118M, T278I, P332A, and V370A) are located near the barbed end of the monomer. The actin filament is a polar structure with a "barbed" end and a "pointed" end, and the monomer barbed end, consisting of subdomains 1 and 3, would be located at the barbed end of the filament (23.Holmes K.C. Popp D. Gebhard W. Kabsch W. Nature. 1990; 347: 44-49Crossref PubMed Scopus (1315) Google Scholar). The filament barbed end is the preferred site for monomer addition during filament elongation. It participates in monomer-monomer contacts longitudinally along the actin helix, and it is the region at which a number of proteins that regulate actin polymerization in the cell exert their control. Patients with these mutations begin noticing hearing loss in their early teens to late twenties depending on the mutation. Hearing loss begins with high frequency sounds and progresses to loss of low frequency sounds, with overall hearing decreasing as the patient ages. Understanding the effects of these mutations at the molecular level in the context of the hair cell is hampered by the small size of the organ involved and the inability to obtain samples of the structures from the patients for biochemical studies. Even if one were to introduce the mutations into model animals and to establish cultured hair cell preparations, the amount of material available for biochemical analysis would be limited. Additionally, within the hair cell, based on the known isoactin content, the mutant actin would likely not account for more than about one-third of the total actin in the cell. This would make it even more difficult to directly assess the effects of the mutations on actin function. An attractive model system for studying these mutant actins is the budding yeast Saccharomyces cerevisiae. Yeast actin is 91% identical to non-muscle γ-actin, and it is encoded by a single gene, ACT1 (24.Ng R. Abelson J. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3912-3916Crossref PubMed Scopus (348) Google Scholar). More important, all six of the residues at which the deafness mutations occur in γ-actin are identical in yeast actin. Many of the actin-binding proteins present in mammalian cells are also found in yeast (25.Drubin D.G. Cell Motil. Cytoskeleton. 1990; 15: 7-11Crossref PubMed Scopus (35) Google Scholar, 26.Bretscher A. Drees B. Harsay E. Schott D. Wang T. J. Cell Biol. 1994; 126: 821-825Crossref PubMed Scopus (59) Google Scholar), and yeast actin will interact with many of the mammalian isoforms of these actin-binding proteins (27.Chen W. Wen K.-K. Sens A.E. Rubenstein P.A. Biophys. J. 2006; 90: 1308-1318Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 28.Wen K.-K. Rubenstein P.A. J. Biol. Chem. 2005; 280: 24168-24174Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The wild-type (WT) 2The abbreviations used are: WT, wild-type; ϵ-ATP, 1,N6-ethenoadenosine 5′-triphosphate; DAPI, 4′,6-diamidino-2-phenylindole; GFP, green fluorescent protein.2The abbreviations used are: WT, wild-type; ϵ-ATP, 1,N6-ethenoadenosine 5′-triphosphate; DAPI, 4′,6-diamidino-2-phenylindole; GFP, green fluorescent protein. actin gene can easily be replaced with a mutant actin gene, generated by site-directed mutagenesis, providing a system in which the mutant actin is the only actin in the cell (29.Shortle D. Novick P. Botstein D. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 4889-4893Crossref PubMed Scopus (112) Google Scholar). The effects of these mutations on actin function within the yeast cell can be assessed by a number of cytological assays (30.McKane M. Wen K.-K. Boldogh I.R. Ramcharan S. Pon L.A. Rubenstein P.A. J. Biol. Chem. 2005; 280: 36494-36501Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). The actins can then be purified and analyzed biochemically to determine the effects each mutation has on actin function in vitro. Finally, one can try to correlate the effects observed in vitro with the altered actin function in vivo. We have used such a system repeatedly to study actin structure-function relationships to delineate the effects a mutation has on the ability of the actin to polymerize, to interact with actin-binding proteins, and to activate myosin, leading to the generation of contractile force (27.Chen W. Wen K.-K. Sens A.E. Rubenstein P.A. Biophys. J. 2006; 90: 1308-1318Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 28.Wen K.-K. Rubenstein P.A. J. Biol. Chem. 2005; 280: 24168-24174Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 30.McKane M. Wen K.-K. Boldogh I.R. Ramcharan S. Pon L.A. Rubenstein P.A. J. Biol. 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Materials—DNase I (grade D) was purchased from Worthington. DE52 DEAE-cellulose was obtained from Whatman. Micro Bio-Spin P-30 Tris columns and Affi-Gel 10-activated resin were purchased from Bio-Rad. ATP was from Sigma. 1,N6-ethenoadenosine 5′-triphosphate (ϵ-ATP), rhodamine-phalloidin, FM 4-64, and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Molecular Probes. The QuikChange® site-directed mutagenesis kit was from Stratagene, and oligodeoxynucleotides were purchased from Integrated DNA Technologies. Yeast cakes for WT actin preparations were purchased from a local bakery. All other chemicals were reagent-grade quality. Construction of Mutant Yeast Strains and Determination of Growth Characteristics—Mutations were introduced into the centromeric plasmid pRS314 (36.Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar) containing the yeast actin coding sequence driven by the ACT1 promoter using the QuikChange® mutagenesis kit according to the manufacturer's instructions. Plasmids containing the desired mutations were introduced into a recipient yeast strain containing a deleted chromosomal ACT1 gene and a plasmid expressing WT actin (pCENWT) as described previously (37.Cook R.K. Sheff D.R. Rubenstein P.A. J. Biol. Chem. 1991; 266: 16825-16833Abstract Full Text PDF PubMed Google Scholar). Plasmid shuffling yielded viable haploid strains for each of the mutations. The plasmids containing the mutant constructs were re-isolated from these strains and sequenced to confirm the presence of the desired mutation. The ability to grow in complete liquid YPD medium (1% yeast extract, 2% peptone, and 2% dextrose) was determined by diluting an overnight culture of each mutant strain into fresh medium at an initial A600 of 0.1 and following growth at 30 °C with agitation. Growth was assessed until the cultures had reached stationary phase. Temperature-sensitive growth was assessed by plating four serial 10-fold dilutions of the cultures on YPD plates, followed by incubation at 24, 30, or 37 °C. Colony size was assessed as a function of time. The ability to grow on glycerol as the sole carbon source was assessed in a similar fashion, except that the cells were plated on YPG medium (YPD medium with the dextrose replaced with 2% glycerol), and the plates were then incubated at 30 °C. Cytology—For observance of cell structures, images were collected with a Zeiss Axioskop 2 Plus microscope using a Plan-Apochromat 100 × 1.4 numerical aperture objective lens and a Spot RT cooled CCD camera (Diagnostic Instruments, Sterling Heights, MI). Camera control and image enhancement were performed using MetaMorph Version 4.5 software (Universal Image Corp., Downingtown, PA). Presentation of cell images was done using Adobe Photoshop. All cellular statistical analysis was based on cell counts of >100 for each sample. Mitochondria in living cells were visualized using a fusion protein in which green fluorescent protein (GFP) was fused to the mitochondrial signal sequence of citrate synthase. The construct used for expression of this protein was kindly provided by Dr. Liza A. Pon (38.Fehrenbacher K.L. Yang H.C. Gay A.C. Huckaba T.M. Pon L.A. Curr. Biol. 2004; 14: 1996-2004Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Cells expressing the plasmid were grown to an A600 of 0.3–0.5 in Ura– synthetic medium to force retention of the URA3-marked plasmid in the otherwise Ura3– cells. An aliquot of cells was resuspended in VECTASHIELD mounting medium (Vector Laboratories, Burlingame, CA), and the cells were then observed by fluorescence microscopy as described above. For mitochondria, ∼40 z-sections were obtained at 0.15-μm intervals through the entire cell. Out-of-focus light was removed by deconvolution using MetaMorph software, and each series of deconvolved images was further rendered with NIH Image J. The actin cytoskeleton was visualized by fluorescence microscopy after staining fixed cells with rhodamine-phalloidin as described previously (30.McKane M. Wen K.-K. Boldogh I.R. Ramcharan S. Pon L.A. Rubenstein P.A. J. Biol. Chem. 2005; 280: 36494-36501Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Vacuoles were observed following exposure of the cells to the dye FM 4-64 as described previously (39.Vida T.A. Emr S.D. J. Cell Biol. 1995; 128: 779-792Crossref PubMed Scopus (1132) Google Scholar). Samples were visualized by fluorescence microscopy as described above. Nuclear and mitochondrial DNAs were visualized following staining of the cells with DAPI as described previously (30.McKane M. Wen K.-K. Boldogh I.R. Ramcharan S. Pon L.A. Rubenstein P.A. J. Biol. Chem. 2005; 280: 36494-36501Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). To measure cell size, mounted samples were visualized by differential interference contrast microscopy. The long axis of the cell was measured using Image J. The average length for each strain was complied from measuring >100 cells for each sample. Actin Biochemistry—WT and mutant actins were purified from lysates of frozen cells using a combination of DNase I-agarose affinity chromatography, DEAE-cellulose chromatography, and polymerization/depolymerization cycling as described previously (40.Cook R.K. Blake W.T. Rubenstein P.A. J. Biol. Chem. 1992; 267: 9430-9436Abstract Full Text PDF PubMed Google Scholar). Quality of the preparations was assessed using SDS-PAGE and Coomassie Blue staining of the gels. The concentration of G-actin was determined from the absorbance at 290 nm using an extinction coefficient of 0.63 ml·mg–1·cm–1. All actins were used within 4 days following completion of purification. Actin was polymerized by the addition of 2 mm MgCl2 and 50 mm KCl to a G-actin sample in a total volume of 120 μl. Polymerization was monitored at 25 °C by following the increase in light scattering of the sample in a thermostatted microcuvette in a FluoroMax-3 fluorescence spectrometer (HORIBA Jobin Yvon Inc.). All polymerization experiments were performed at least three times with different actin preparations. Dynamic light scattering measurements of the G-actin samples of V370A and WT actins were performed at several different concentrations ranging from ∼0.25 to 10 μm at 25 °C using a DynaPro dynamic light scattering instrument containing a temperature-controlled microsampler (Protein Solutions, Inc.). Determination of the particle size for each sample was calculated using Dynamic Version 5.26.38 (included with the instrument), and the average particle size at each concentration was based upon measuring three independent samples. The apparent melting temperatures of WT and mutant actins were determined using circular dichroism by following the change in ellipticity of the G-actin sample at 222 nm as a function of temperature between 25 and 90 °C as described previously (35.Yao X. Nguyen V. Wriggers W. Rubenstein P.A. J. Biol. Chem. 2002; 277: 22875-22882Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Measurements were made on an Aviv 62 DS spectropolarimeter. Data were fit to a two-state model, and the apparent Tm value was determined by fitting the data to the Gibbs-Helmholtz equation to approximate the temperature at which 50% of the actin was denatured. The ability of G-actin to exchange its bound nucleotide was assessed by first loading the actin with ϵ-ATP and following its displacement from the actin in the presence of a large excess of ATP as described previously (35.Yao X. Nguyen V. Wriggers W. Rubenstein P.A. J. Biol. Chem. 2002; 277: 22875-22882Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Exchange rates were determined by fitting the data to a single exponential expression using BioKine Version 3.1. To visualize actin filaments, samples of 2.4 μm F-actin were deposited onto carbon-coated Formvar grids, negatively stained with 1% uranyl acetate, and observed with a JOEL 1230 transmission electron microscope (University of Iowa Central Electron Microscopy Facility). Lengths of individual filaments for WT and V370A actins were measured using Image J. Effect of Mutations on the Rate and Extent of Cell Growth—We first examined the cells for global effects of the mutations on cell behavior by assessing their ability to grow on YPD plates at different temperatures. All six strains were temperature-sensitive for growth at 37 °C (data not shown). We then examined more quantitatively their ability to grow in complete YPD medium at 30 °C. The results revealed that the mutations group themselves into two different growth categories (Fig. 2). Cells carrying the T89I and P264L mutations grew slower than WT cells and to a final density of ∼70% that achieved by WT cells. The second group, consisting of K118M, T278I, P332A, and V370A, exhibited significantly slower rates of logarithmic growth and leveled off at a density of ∼30% that of WT cells. One possible reason for decreased growth of this magnitude is a disruption of proper mitochondrial function caused by the mutation. Yeast requires actin for mitochondrial inheritance, maintenance of mitochondrial morphology (38.Fehrenbacher K.L. Yang H.C. Gay A.C. Huckaba T.M. Pon L.A. Curr. Biol. 2004; 14: 1996-2004Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 41.Boldogh I.R. Fehrenbacher K.L. Yang H.C. Pon L.A. Gene (Amst.). 2005; 354: 28-36Crossref PubMed Scopus (46) Google Scholar, 42.Simon V.R. Karmon S.L. Pon L.A. Cell Motil. Cytoskeleton. 1997; 37: 199-210Crossref PubMed Scopus (117) Google Scholar, 43.Yang H.C. Simon V. Swayne T.C. Pon L. Methods Cell Biol. 2001; 65: 333-351Crossref PubMed Google Scholar), and stabilization of mtDNA. A single actin mutation (H372R) we recently studied disrupts all three of these processes (30.McKane M. Wen K.-K. Boldogh I.R. Ramcharan S. Pon L.A. Rubenstein P.A. J. Biol. Chem. 2005; 280: 36494-36501Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Mitochondrial function may be easily assessed by examining the ability of the cells to grow on glycerol as the sole carbon source. A mitochondrial glycerol-3-phosphate dehydrogenase is required for conversion of glycerol 3-phosphate to dihydroxyacetone phosphate, which can then be further metabolized by glycolysis. Mitochondrial defects often result in elimination of this activity. Fig. 3 shows that, although cells carrying the T89I and P264L mutations grew on glycerol, those carrying the four remaining mutations (K118M, T278I, P332A, and V370A), which exhibited the most severe growth retardation in complete medium, could not. Effect of the Mutations on Actin Cytoskeletal Patterns and Mitochondrial Morphology—Mitochondrial inheritance requires the movement of these organelles from the mother cell to the bud along polarized actin cables running between these cells. The current theory for mitochondrial movement is that anterograde movement is Arp2/3 complex-mediated, whereas the myosin Myo2p is necessary for retention of the organelle in the bud (42.Simon V.R. Karmon S.L. Pon L.A. Cell Motil. Cytoskeleton. 1997; 37: 199-210Crossref PubMed Scopus (117) Google Scholar, 44.Simon V.R. Swayne T.C. Pon L.A. J. Cell Biol. 1995; 130: 345-354Crossref PubMed Scopus (167) Google Scholar, 45.Boldogh I.R. Yang H.C. Nowakowski W.D. Karmon S.L. Hays L.G. Yates III, J.R. Pon L.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3162-3167Crossref PubMed Scopus (147) Google Scholar, 46.Boldogh I.R. Ramcharan S.L. Yang H.C. Pon L.A. Mol. Biol. Cell. 2004; 15: 3994-4002Crossref PubMed Scopus (85) Google Scholar). A properly polarized actin cytoskeleton is also necessary for control of the fission/fusion events that regulate mitochondrial morphology (42.Simon V.R. Karmon S.L. Pon L.A. Cell Motil. Cytoskeleton. 1997; 37: 199-210Crossref PubMed Scopus (117) Google Scholar). Besides actin cables, actin patches (a second form of polymerized actin) are also observed. These are sites of endocytosis (47.Engqvist-Goldstein A.E. Drubin D.G. Annu. Rev. Cell Dev. Biol. 2003; 19: 287-332Crossref PubMed Scopus (493) Google Scholar), and their distribution is cell cycle-dependent. At early stages following initial enlargement of the bud, the patches are confined almost entirely to the growing bud, whereas at later times, the patches are evenly distributed between the bud and mother cell (48.Amberg D.C. Mol. Biol. Cell. 1998; 9: 3259-3262Crossref PubMed Scopus (82) Google Scholar). Disruption of the actin cytoskeleton by actin mutations may lead to depolarized isotropic growth of the cell, resulting in larger and rounder cells. Decreased cell size is often observed in mitochondrion-deficient strains, reflecting their inability to generate enough energy to support optimal growth.
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