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Loss of Spatial Control of the Mitotic Spindle Apparatus in a Chlamydomonas Reinhardtii Mutant Strain Lacking Basal Bodies

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Loss of Spatial Control of the Mitotic Spindle Apparatus in a Chlamydomonas Reinhardtii Mutant Strain Lacking Basal Bodies Copyright 0 1995 by the Genetics Society of America Loss of Spatial Control of the Mitotic Spindle Apparatus in a Chlamydomonas reinhardtii Mutant Strain Lacking Basal Bodies Linda L. Ehler, Jeffrey A. Holmes' and Susan K. Dutcher Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309-0347 Manuscript received April 27, 1995 Accepted for publication August 4, 1995 ABSTRACT The bld2-1 mutation in the green alga Chlamydomonas reinhardtii is the only known mutation that results in the loss of centrioles/basal bodies and the loss of coordination between spindle position and cleavage furrow position during cell division. Based on several different assays, bld2-1 cells lack basal bodies in >99% of cells. The stereotypical cytoskeletal morphology and precise positioning of the cleavage furrow observed in wild-type cellsis disrupted in bld2-1 cells. The positionsof the mitotic spindle and of the cleavage furrow are not correlated with respect to each other or with a specific cellular landmark during cell division in bld2-1 cells. Actin has a variable distribution during mitosis in bld2-1 cells, but this aberrant distribution is not correlated with the spindle positioning defect. In both wild- type and bld2-1 cells, the position of the cleavage furrow is coincident with a specialized set of microtu- bules found in green algae knownas the rootlet microtubules.We propose that the rootlet microtubules perform the functionsof astral microtubules and that functional centrioles are necessarythe organiza- for tion of the cytoskeletal superstructure critical for correct spindle and cleavage furrow placement in Chlamydomonas. HE position of the mitotic spindle apparatus must Physarum and Naegleria cells do not have centrioles T be coordinated bothtemporally and spatially with during mycelial growth (FULTONand DINGLE1971), the cleavage furrow to ensure correct partitioning of and higher plant cells do not have centrioles. In addi- the cellular contents.Incorrect placement of these tion, the Drosophila haploid tissue culture cell line structures with respect to each other would result in 1182-4, which was isolated from dead embryos of the cellular catastrophe. The elaborate mechanisms that female sterile strain mh1182, lacks centrioles when ex- exist for a cell to monitor the correct replication and amined by serial thin-section electron microscopy. The segregation of itsDNA must act in concertwith cleavage cell line undergoes growth and mitotic divisions, but it furrow positioning. Furthermore, correct segregation spontaneously diploidizes, has a high levelof aneu- of developmental determinants and implementation of ploidy and contains large numbers of multinucleate developmental programs may require precise cleavage cells (DEBEC et al. 1982; DEBEC1984; SZOLLOSIet aZ. furrow position within cells undergoing development. 1986). It is unclear whether these defects arise from For example, inCaenarhabditis elegans, the specific orien- the lack of centrioles or from the haploid/aneuploid tation of the cleavage furrow is necessary for the correct nature of the cell line. In contrast,when the centrioles localization of P granules to only one daughter cell in and most of the pericentriolar material are surgically thesubsequent divisionof the P1 cell (HYMANand removed from cells of the monkey cell line BSG1, an WHITE1987). organized array of microtubules is regenerated but cen- The mitotic spindles of many cells are nucleated by trioles are not, and the cells do not complete cell divi- the centrosomes, which contain a pair of centrioles/ sion (MANIOTIS and SCHLIWA1991). It is unclear basal bodies andamorphous pericentriolar material whether the inability of these surgically altered cells to (PCM) that usually nucleates amicrotubule array divide is due to a lack of centrioles or to a missing (GOULDand BORIS 1977). Although centrosomes ap- component(s) of the PCM. The evidence for the role pear to be essential for formation of the spindle and of centrioles in cell division remains inconclusive. the mitotic asters, it is unclear whether the centrioles The exact mechanisms involved in positioning the are necessary for this function. Mouse meiotic spindles mitotic apparatus are unknown. One current model is do not have centrioles (CALARCO-GILLAMet al. 1983), that the centrosomes migrate to a position previously established or maintained by a microfilament network. Corresponding authm: Susan K. Dutcher, Department of Molecular, The interaction with the actin cytoskeleton is proposed Cellular, and Developmental Biology, University of Colorado, Boul- to occur via the astral microtubules that radiate from der, CO 80309-0347. E-mail: [email protected] the centrosomes (HYMANand WHITE 1987; PALMERet 'Present address: Intercampus Program, Molecular Parasitology, 3333 California Street University of California, San Francisco, California, al. 1992).In the P blastomeres of C. elegans, the 941431204, centrosomes separate and migrate to positions directly Genetics 141: 945-960 (November, 1995) 946 L. L. Ehler, J. A. Holmes and S. K. Dutcher opposite each other and subsequently both rotate 90 formation is dependent on microtubules; loss of micro- deg to align on the anterior-posterior axis. Rotation of tubules from treatment with colchicine before midana- the centrosomes shows sensitivityto microtubuleinhibi- phase prevents cleavage furrow initiation (BWS and tors (HYMANand WHITE 1987). Laser irradiation be- EVANS1940; HAMAGUCHI 1975).These results suggest tween the centrosome and the cell cortex results in that theasters and probably the astral microtubules play the cessation of centrosomal rotation and implicates a a key role in positioning the cleavage furrow. particular cortical site in the rotational events (HYMAN We have characterized a C. reinhardtii mutant strain, 1989). Treatmentof P blastomeres with cytochalasin D, bld2-I, that lacks centrioles/basal bodies. The strain was an inhibitor of microfilament assembly, results in the originally isolated by GOODENOUGH and ST. CLAIK failure of centrosomes to rotate (HILLand STROME (1975), has a single genetic lesion, and represents the 1988, 1990; HYMAN1989). Actin and actin capping pro- first allele at a previously unidentified locus. Cells lack tein have been shown to transiently accumulate at a centrioles/basal bodies in >99% of the population and cortical site that also has astral microtubules in its vicin- GOODENOUCHand ST. CWR (1975) proposed that the ity during thetime of centrosomal rotation;blastomeres BLD2 gene product is required for the formation of that do not undergo centrosomal rotation do notaccu- doublet and triplet microtubules. In addition to the mulate capping protein atthis site (WADDLEet al. 1994). basal body defect, we have found that cellular organiza- In the budding yeast, Saccharomyces cerevisicce, charac- tion is disrupted. The placement of the mitotic spindle terization of strains that have abnormal placement of is no longer correlated spatially or temporally with the the mitotic apparatus has implicated astral microtu- cleavage furrow and is misplaced relative to the sterec- bules, cytoplasmic dynein, dynactin complex, and actin typed cytological position seen in wild-typecells. The microfilaments in establishing or maintaining the spin- position of the cleavage furrow is also aberrant, but it dle in the correct position before anaphase. At the re- always colocalizes with specialized structures known as strictive temperature, the o-tubulin mutant straintub2- rootlet microtubules. The rootlet microtubules in bld2- 401 loses astral microtubules preferentially, the mitotic 1 cells are disorganized and often mispositioned in rela- spindles misorient; anucleate and multinucleate cells tion to the mitotic spindle. These observations clearly accumulate (HUFFAKERet al. 1988; SULLIVANand HUE implicate the BLD2 gene product as a component in- FAKER 1992). Deletion mutations in the cytoplasmic volved in the correct positioning of the mitotic spindle. dynein heavy chain gene DHCI (DYNl) orflM1 result The role of the BLD2 gene product may be in the initia- in minor spindle orientation defects (ESHELet al. 1993; tion or structure of functional centrioles, which may be LI et al. 1993; MCMILLANand TATCHELL1994). Spindle necessary to maintain the elaborate cytoskeletal organi- misorientation in dhcl (dynl) disruption strains is ac- zation critical for correct cell division in C. reinhardtii. companied by elongated astral microtubules and an increase in cells that are binucleate. Act5 is a member MATERIALS AND METHODS of the actin-related protein family Arpl and is homolo- Strains and culture conditions: The 137c mt+ strain of C. gous to the actin-like protein found in the vertebrate reinhardtii was used as the wild-type strainfor this study unless dynactin complex. Deletions of the ACT5 gene result otherwise noted. The bldl-l and the bld2-1 strains were origi- in minor spindle placement defects (GILL et al. 1991; nallyisolated by GOODENOUGHand Sr. CIAIR (1975) and MUHUAet al. 1994). obtained from the Chlamydomonas Genetics Center. The Several lines of evidence suggest that the position of aflagellate strain 426 was isolated in a screen for cells that could not swim after heat shock treatment (42", 30 min) of the cleavage furrow is determined by the position of a member of the F, generation from a cross of strain 137c to the mitotic apparatus. The furrow always forms at right the polymorphic strain, CC1952 (GROSSet al. 1988).Cultures angles
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