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649 Development 126, 649-658 (1999) Printed in Great Britain © The Company of Biologists Limited 1999 DEV0193 glsA, a Volvox gene required for asymmetric division and germ cell specification, encodes a chaperone-like protein Stephen M. Miller and David L. Kirk* Department of Biology, Washington University, St. Louis, MO, 63130, USA *Author for correspondence (e-mail: [email protected]) Accepted 4 December 1998; published on WWW 20 January 1999 SUMMARY The gls genes of Volvox are required for the asymmetric divisions that set apart cells of the germ and somatic lineages during embryogenesis. Here we used transposon tagging to clone glsA, and then showed that it is expressed maximally in asymmetrically dividing embryos, and that it encodes a 748-amino acid protein with two potential protein-binding domains. Site-directed mutagenesis of one of these, the J domain (by which Hsp40-class chaperones bind to and activate specific Hsp70 partners) abolishes the capacity of glsA to rescue mutants. Based on this and other considerations, including the fact that the GlsA protein is associated with the mitotic spindle, we discuss how it might function, in conjunction with an Hsp70-type partner, to shift the division plane in asymmetrically dividing cells. INTRODUCTION and Quatrano, 1993; Guo and Kemphues, 1996; Kraut et al., 1996; McGrail and Hays, 1997; Skop and White, 1998; Waddle et al., 1994; White and Strome, 1996; Zwaal et al., 1996). However, we still are far from having any sort of overview of the range of mechanisms that may be involved in regulating division symmetry. The spherical multicellular alga Volvox carteri has features that make it appealing as an additional, and conceivably simpler, model for studying the control of division symmetry. The two cell types of the Volvox spheroid (mortal somatic cells and immortal germ cells) are set apart during embryogenesis by a highly regular pattern of visibly asymmetric divisions, and there is no indication that these quantitatively asymmetric divisions are complicated by any qualitative asymmetry. Indeed, several lines of evidence indicate that in V. carteri it is just the amount, and not the quality, of cytoplasm that a cell possesses at the end of cleavage that determines whether it will activate the germinal or somatic pathway of gene expression and cytodifferentiation (Kirk et al., 1993). An asexual V. carteri adult consists of approximately 2000 small, biflagellate somatic cells located at the surface, and approx. 16 large, asexual reproductive cells (‘gonidia’) located just below the surface of a transparent glycoprotein-rich sphere (Kirk, 1998; Starr, 1970). Each mature gonidium produces an embryo containing all the cells that will be present in an adult of the next generation by initiating a program of rapid cleavage divisions, a stereotyped subset of which are visibly asymmetric and set apart large/small sister-cell pairs that function as gonidial and somatic initials, respectively. At the end of cleavage the gonidial initials are approximately three times the diameter of somatic initials. Studies to date all indicate that it Precise positioning of cell division planes plays a crucial role in the development of many organisms, by determining the sizes, shapes, cytoplasmic compositions, and/or spatial relationships of the daughter cells. Cell-division geometry is particularly important in those cases in which cytoplasmic determinants are asymmetrically distributed inside the predivisional cell, or when potential sources of fatedetermining signals are asymmetrically distributed outside of it (Horvitz and Herskowitz, 1992). As a rule, developing plants (in which readjustment of cellular relationships via cell migration is not an option) are even more dependent on defined and regular patterns of cell division geometry than developing animals are. In most eukaryotes the plane of cell division can be predicted from the position of the mitotic spindle. However, in animals it is the positions occupied by centrosomes at prophase that determine the orientation of both the spindle and (via the polar asters that they organize) the division plane (Raff and Glover, 1989; Strome, 1993), and in plants it is the ‘preprophase band’ of circumferential microtubules that predicts the orientation of both the spindle and the division plane (Lloyd, 1991), but in few cases do we have a detailed understanding of how the locations of centrosomes or preprophase bands are specified at critical stages of development. In recent years significant progress has been made in identifying gene products in various model organisms that participate at some level in determining the geometry and/or consequences of particular embryonic cell divisions (Broadus and Doe, 1997; Di Laurenzio et al., 1996; Doe, 1996; Goodner Key words: Cell division, Green algae, Hsp40, J protein, Volvox carteri 650 S. M. Miller and D. L. Kirk is this size difference that is critical for deciding cell fate: cells >8 µm at the end of cleavage – no matter where or how they have been formed – always develop into gonidia, whereas smaller cells always become somatic cells (Kirk et al., 1993). Thus, it is clear that cell division symmetry plays a central role in V. carteri development. Mutational analysis has revealed a class of genes (gonidialess, gls) whose products are required for asymmetric, but not for symmetric, cell division (Kirk et al., 1991). In Gls mutants all cell divisions are symmetric, and fully cleaved embryos contain only small blastomeres that differentiate as somatic cells. These mutations can only be recovered and maintained in strains having the Regenerator (Reg) phenotype, in which somatic cells are able to redifferentiate as functional germ cells (see Kirk et al., 1999). As a first step toward learning how the gls loci act to control asymmetric division in V. carteri, we have used transposon tagging to identify and recover and characterize the glsA gene, and have found that it encodes a protein with a chaperone domain (the J domain) that must be functional for the GlsA protein to play its essential role in asymmetric cell division. MATERIALS AND METHODS Volvox strains and cultivation conditions The standard Volvox strains, media and culture conditions used, the methods used for inducing and isolating putative transposon-tagged mutants, for nuclear co-transformation, and selection of Nit+ mutants were all as described by Kirk et al. (1999). The Gls mutants studied here were all isolated from 24°C cultures of strain 22reg1 (a stable Reg derivative of CRH22) and screened for their ability to generate Gls+ revertants when grown at 32°C and/or 24°C. DNA methods Volvox genomic DNA was prepared as described by Miller et al. (1993) and then further purified by spermine precipitation and phenol/chloroform extraction. Southern blot analysis was as previously described by Miller et al. (1993) and DNA sequencing was as described by Kirk et al. (1999). A partial genomic library of gel-purified BamHI restriction fragments of strain 22gls1 DNA in pBluescript KS+ (KS+; Stratagene) was screened with a Jordan probe, yielding a plasmid, B3.4-1, that contained the novel 3.4-kb BamHI restriction fragment of strain 22gls1. Phage λ clones bearing glsA sequence were then isolated by screening an existing Volvox genomic library (Mages et al., 1988) with a probe derived from B3.4-1, according to Sambrook et al. (1989). Plasmids LV304 and LV307 were constructed by subcloning the 8.3kb SalI and 9.5-kb XhoI fragments of λglsA4, respectively, into KS+. Plasmid LV377 was constructed by replacing the 0.1-kb EcoRVBstEII fragment from the 5′ end of the LV304 insert with the 1.6-kb EcoRV-BstEII 5′ glsA fragment of LV307. Plasmid LV399, which contains a variant of the LV377 insert, in which the region encoding the conserved HPD tripeptide of the GlsA J domain has been to mutated to encode QPA, was generated as follows. Plasmid LV300-2′ was synthesized by a PCR utilizing two 45-mers, pMut1 (5′AGACATGTCTGGAGAACCAGCCGGCTAAGGCCCTCATTAATGTCA3′) and pMut2 (5′TCCGGTAGGCCGCCCTGATCTGGGCCTCGGAAGCGGTCCACCGCT3′) as primers and the 3.6-kb plasmid LV300-2 as template. LV300-2 contains the 650-bp BamHI-ApaI fragment of glsA (Fig. 3) cloned into KS+. Primer pMut1 contains 2 nucleotide mismatches with respect to its glsA target sequence, so that successful full-length amplification of the plasmid, followed by ligation of the fragment ends to each other, created a plasmid identical to LV300-2 except at the position of the two nucleotide mismatches. The insert of LV300-2′ was sequenced to verify that the two mismatches directed by pMUT1, and no other mutations, were incorporated into glsA sequence. Plasmid LV399 was generated by replacing the corresponding portion of plasmid LV377 with the mutated version from LV300-2′. Plasmid LV388, with an insert encoding a GlsA variant tagged with the influenza hemagglutinin (HA) epitope (Atassi and Webster, 1983; Field et al., 1988), was generated as follows. Oligonucleotides pHA1 (5′GTCACCTACCCGTACGACGTCCCGGACTACGCC3′) and pHA-2 (5′GTGACGGCGTAGTCCGGGACGTCGTACGGGTAG3′) were annealed (Sambrook et al., 1989) to form small double-stranded DNA fragments having BstEII compatible ends. These were ligated to BstEII-digested LV377, and recombinant plasmids that contained both a regenerated BstEII restriction site and a novel AatII restriction site were sequenced to determine the number of inserts present and to verify that the reading frame was intact. RT-PCR and semi-quantitative RT-PCR RNA samples were isolated as described by Kirk and Kirk (1985) from somatic cells, gonidia or embryos isolated from synchronous cultures at the developmental stages of interest and were then used to generate first-strand cDNAs using a reverse-transcriptase kit (Stratagene). PCRs used primers gls32 (5′CATCCTTAGCGACCCAGCCAAG3′) and gls33 (5′GCCGCCTCCACGTCCTCCTC3′) to generate a 370-bp glsA-specific product, and primers c38-1 and c382 (Kirk et al., 1999) to generate the 355-bp S18-specific (ribosomal protein-encoding) product that served as a control. Production, electrophoretic separation, and quantitation of PCR products were all as described by Kirk et al. (1999). Protein-accumulation studies with HA-tagged GlsA Strain 22gls1-NRG was isolated as a Nit+ Reg+ Gls+ derivative of 22gls1 following co-transformation with three plasmids carrying the nitA gene, the wild-type regA gene (Kirk et al., 1999), and the gene encoding the HA-tagged version of GlsA. Extracts of 22gls1-NRG gonidia, embryos and somatic cells that had been isolated at selected developmental stages by the methods of Tam and Kirk (1991) and then prepared for and subjected to SDS-PAGE by standard methods (Sambrook et al., 1989) were transferred to nitrocellulose membranes (Micron Separations, Inc., Westborough, MA) for western blot analysis. SuperSignal® substrate (Pierce Biochemicals, Rockford, IL) was used to detect western signals according to the manufacturer’s recommendations, using 10% nonfat dried milk as the blocking agent. Primary antibody (anti-HA monoclonal 12CA5, provided by L. Ellis, Washington Univ.) was diluted 1:500 in blocking solution, and secondary antibody (goat anti-mouse:horseradish peroxidase conjugate; Biorad Laboratories, Melville, NY) was diluted 1:5,000 in blocking solution. All aspects of the preparation of 22gls1-NRG samples for indirect immunofluorescent examination were as described by Kirk et al. (1999), except that primary antibody labeling was with a mixture of a 1:10 dilution of the 12CA5 monoclonal antibody and a 1:1,000 dilution of a rabbit polyclonal to Chlamydomonas β-tubulin (kindly contributed by Joel Rosenbaum’s laboratory) and the secondary antibody labeling was with a mixture of Cy™2-labeled goat antirabbit IgG and Cy™3-labeled goat anti-mouse IgG (Amersham), both at 2 µg/ml. RESULTS Identification and characterization of a candidate transposon-tagged Gls mutant Two attributes of Jordan facilitate its use as a gene-tagging agent: (i) its transposition can be induced 30-fold or more by The Volvox glsA gene mild environmental stresses (such as cultivation at 24°C, which is near the lower limit for growth of V. carteri; S. M. Miller, unpublished observations), and (ii) many mutations caused by Jordan insertions are revertible (Miller et al., 1993, plus unpublished observations). When replicate cultures of the regA−, nitA− strain 22reg1 were grown for several generations at 24°C and screened microscopically, 24 independent Gls mutants were recovered. Five of these were highly revertible, and one, 22gls1, reverted much more frequently at 24°C than at 32°C, indicating that it was an excellent candidate to harbor a Jordan insertion in a gls gene. In common with about 10% of >100 Gls mutants that we have isolated, 22gls1 exhibits a slightly leaky (‘quasi-Gls’) phenotype: approximately half of all 22gls1 spheroids contain zero or one gonidia, most of the rest contain two or three gonidia, individuals with four gonidia are rare, and all spheroids, regardless of the number of gonidia they themselves contain, generate progeny with zero to four gonidia in similar frequencies. 22gls1 also has a cold-sensitive growth defect. At 32°C it grows as vigorously as its Reg progenitor, its Gls+ revertants, and other GlsReg strains. However, at 24°C 22gls1 grows far more slowly than any of those other strains, never completes more than one life cycle, and soon bleaches and becomes moribund. Systematic studies have failed to identify a single cold-sensitive phase: cells within a single spheroid cease growing and bleach in a wholly asynchronous, stochastic manner. The only robustly growing spheroids that appear in 22gls1 cultures at 24°C are ones that have undergone a stable reversion to Gls+. The fact that the Gls phenotype of 22gls1 and its cold-sensitive growth defect invariably co-revert provides strong evidence that they result from the same genetic lesion. Microscopic analysis of cleaving 22gls1 embryos confirmed that the Gls phenotype results from a defect in asymmetric cell division at 32°C: embryos that have produced one to four large cells by asymmetric divisions are seen with the same frequency as adults with that number of gonidia (Fig. 1 and data not shown). Isolation of the glsA gene DNA blots probed with Jordan revealed a 3.4-kb BamHI fragment in 22gls1 DNA that was not present in DNA from 22reg1 or any of several Gls+ revertants tested (data not shown). Repeated loss of the novel Jordan-containing fragment of 22gls1 in conjunction with reversion of the Gls phenotype provided strong evidence that those two traits were causally related. Therefore, the novel 3.4-kb fragment was cloned, yielding the plasmid B3.4-1, and genomic DNA flanking the transposon was isolated. When this genomic DNA was used to probe a blot containing BamHIdigested DNAs of 22reg1, 22gls1, and six independent Gls+ revertants of 22gls1, the fragment detected in 22gls1 was found to be larger by 1.6 kb (the size of Jordan, Miller et al., 1993) than it was in 22reg1 or any of the six revertants (Fig. 2A). This result provided strong evidence that we had 651 cloned the copy of Jordan that is responsible for the Gls phenotype of strain 22gls1, and that the DNA flanking Jordan in B3.4-1 represented a portion of the gls locus of interest. Genomic clones that hybridized with fragments isolated from B3.4-1 were tested for their ability to rescue the Gls phenotype of 22gls1 by biolistic co-transformation, using the nitA (nitrate-reductase encoding) gene as the selectable marker (Schiedlmeier et al., 1994). A Gls+ Nit+ strain recovered after bombardment with λglsA4 was found to possess both the 3.4kb BamHI fragment representing the Jordan-inactivated version of the gene, and a 1.8-kb fragment corresponding to the wild-type allele (Fig. 2B), demonstrating that its Gls+ phenotype was the result of transformation rescue, not reversion of the transposon-induced mutation. This, in turn, proved that λglsA4 contains a functional copy of the gls gene of interest. We call this gene glsA. The boundaries of glsA were defined more closely when several subclones derived from λglsA4 and a longer clone, λglsA5, were tested for their ability to rescue the Gls phenotype of 22gls1 by transformation and it was shown that clones LV304, LV307, and LV377 were capable of such rescue, but LV322 was not (Fig. 3B). However, plasmid LV377, which contains an insert extending to both the right and left of the region of overlap of the LV304 and LV307 inserts, was found to be >15 times as efficient at rescuing the Gls phenotype than LV304 and LV307 were (67% vs. approx. 4% cotransformation, respectively). The source of this discrepancy was discovered when sequencing of the glsA cDNA was completed and it was established that the region of overlap between LV304 and LV307 contains very little upstream DNA, and does not contain the entire glsA transcription unit (Fig. 3B,C). DNA sequence of the coding region of glsA All attempts to detect a glsA message on northern blots, or to isolate glsA cDNAs from existing Volvox libraries were Fig. 1. The Gls phenotype of strain 22gls1. Representative young adults (A-C) and postcleavage, pre-inversion embryos (D-F), of the wild-type (EVE; A and D), Gls (22gls1; B and E), and revertant (22gls1R4; C and F) strains. Note that the 22gls1 embryo in E contains only one large cell (presumptive gonidium). 652 S. M. Miller and D. L. Kirk Fig. 2. Analysis of revertants and putative transformants of 22gls1. (A) Southern blot of BamHI-digested DNAs from 22gls1, its progenitor (22reg1), and 6 of its Gls+ revertants (R1-R6) probed with P1 (see Fig. 3B). (B) Southern blot of BamHI-digested DNAs from 22reg1, 22gls1, and 5 Gls+ strains recovered following transformation of 22gls1 with λglsA4 (T1), LV304 (T2 and T3), and LV307 (T4 and T5) probed with P1. glsA transcript abundance peaks during early embryogenesis To determine the extent to which glsA message levels are spatially and temporally regulated, we used semi-quantitative RT-PCR to measure the relative abundance of glsA transcripts in somatic cells and gonidia/embryos throughout the life cycle. This revealed that glsA transcript levels in gonidia are low during most of the life cycle, increase sharply at the beginning of embryogenesis, and peak 4 hours later, at a time that coincides roughly with the occurrence of the asymmetric cell divisions at the sixth, seventh and eighth cleavage cycles (Fig. 4A). In contrast, glsA transcript levels were low in somatic cells throughout the life cycle. (The small peak seen in the somatic sample at the time glsA transcript is maximally abundant in embryos (Fig. 4A) could be accounted for if this sample had contained as many as one embryo per 104 somatic cells, which is a level of contamination we can not rule out.) GlsA protein is present and associated with the mitotic spindle during cleavage A transgenic, morphologically wild-type strain (22gls1-NRG) that makes a GlsA protein tagged with the influenza hemagglutinin (HA) epitope was used for a western-blot analysis of stage- and cell-type-specific abundance of GlsA. Extracts of all embryos examined contained substantial amounts of a protein recognized by the anti-HA antibody that migrated as an approx. 100-kDa band on SDS-PAGE (Fig. 4B). This same protein was present, but in greatly diminished quantity, in 24-hour-old juvenile spheroids (which contain both somatic cells and young gonidia), but it could not be detected in somatic cells, in gonidia that had been isolated 2-4 hours prior to the onset of cleavage, or in embryos from a control strain (EVE) not bearing the tagged glsA gene. These results indicate that synthesis of GlsA protein begins in mature gonidia just as they prepare to initiate cell division, and then continues through most of the cleavage period. It is unknown whether the weak signal detected in juveniles reflects continuing synthesis, or just residual GlsA that had been synthesized a day earlier, during embryogenesis. unsuccessful, indicating that the glsA transcripts must be of very low abundance. We therefore sequenced the 8.3-kb SalI fragment from genomic clone LV304, subjected the sequence to BLAST analysis (Altschul et al., 1990) and learned that glsA appeared to encode a protein related to several in the database. When primers designed to amplify this putative coding sequence were used to perform RT-PCR with RNA from cleaving embryos, four overlapping PCR products were produced (Fig. 3C). Sequencing confirmed that these putative glsA PCR products not only encoded the polypeptide that had been detected by BLAST analysis, they appeared to represent the entire coding region of the glsA locus (which consists of 16 exons and 15 introns) and encode a 748-residue, approx. 82-kDa protein (Fig. 3C). The AUG identified in Fig. 3C is almost certainly the authentic initiation codon, because there is an in-frame stop codon 60 bp upstream of it in the cDNA, and the next AUG codon is 474 codons downstream. The codon bias of the open reading frame (ORF) encoding the predicted GlsA protein agrees Jordan 1 kb well with the codon bias of previously * X B R S N A BN C N X A characterized volvocalean ORFs (Schmitt et al., 1992), and no reasonable candidate ORFs Rescue λglsA5 could be detected in the other two reading LV322 + λglsA4 frames. Both the genomic and the cDNA B + LV307 sequences have been submitted to GenBank + LV304 + (accession numbers: AF110134 and LV377 P1 P2 AF110135, respectively). The truncated glsA gene carried on plasmid AUG UAA LV304, which rescues the Gls phenotype of C mutant 22gls1 with low efficiency, contains just 27 bp of 5′ untranslated region. Presumably the Fig. 3. The glsA locus. (A) Map of the locus showing only those restriction sites that truncated gene must utilize cryptic promoter and were used for manipulation of clones discussed in this report. The labeled triangle start site sequences in the plasmid, or become indicates where Jordan is inserted in 22gls1. A, ApaI; B, BamHI; C, ScaI; S, SalI; N, juxtaposed fortuitously to such elements when NgoMI; R, EcoRV; X, XhoI; N*, NgoMI site that was created in the mutant plasmid it integrates into the genome, in order to LV399. (B) Clones used in transformation rescue and DNA blot studies. The region function properly. Plasmid LV307, which of overlap among the clones capable of mutant rescue is indicated by the vertical carries a truncated glsA gene predicted to dashed lines. Cross-hatched boxes indicate fragments (P1 and P2) used to probe encode a protein that is missing its carboxy DNA blots. (C) Exon/intron structure of the glsA gene. Boxes represent proteinterminal 124 amino acids, is also inefficient at coding exons, and inverted carets represent introns. Lines at bottom indicate overlapping fragments of cDNA that were generated by RT-PCR and sequenced. rescue. The Volvox glsA gene Examination of early-cleavage 22gls1-NRG embryos by indirect immunofluorescence with the anti-HA antibody revealed clearly that the antigen is most concentrated in the vicinity of the mitotic spindle in early, symmetrically dividing embryos (Fig. 4D). A smaller quantity of antigen is also detected, however, in a crescent-shaped region of the cytoplasm posterior to (i.e., on the chloroplast side of) the nucleus. (Neither of these staining patterns are seen in control embryos lacking HA-tagged GlsA.) So far, technical difficulties have prevented a detailed analysis of whether the protein is localized differently in asymmetrically vs. symmetrically dividing blastomeres in the 6th, 7th and 8th cleavage cycles. The Jordan insertion in mutant 22gls1 results in aberrantly spliced glsA transcripts Sequencing revealed that in strain 22gls1 Jordan is inserted in the fourth intron of glsA, 15 bp upstream of the fifth exon (Fig. 3A). To test the supposition that insertion of the transposon this close to an intron-exon boundary might interfere with splicing, we used RT-PCR to amplify for sequencing the relevant portion of the glsA transcripts from the wild-type and mutant strains. Whereas a single 370-bp product is detected following a PCR using primers that straddle the site of the Jordan insertion and the wild-type RT template, a pair of products are detected when the mutant RT template is used: a smaller (approx. 300-bp) product that lacks the fifth exon, and a larger (approx. 450-bp) product containing the 370 bp present in the wild-type product, plus 62 bp derived from the transposon and its target-site duplication, and the 15 bp of intron lying between the transposon and the fifth exon. Both aberrant transcripts in the mutant are predicted to encode truncated proteins because of frameshifts. Therefore, the transposon insertion in 22gls1 apparently incapacitates the splice acceptor site at the junction of intron 4 and exon 5, leading to the production of two improperly spliced, defective transcripts. We cannot eliminate the possibility, however, that a small number of properly spliced transcripts are produced, accounting for the quasi-gonidialess phenotype of strain 22gls1. 653 M (Fig. 5B). We will review what is known about the functions of such domains in the Discussion. One of the proteins most similar to GlsA (BLAST probability score approx. 10−52) is MPP11, a human ‘M-phase phosphoprotein’ that is phosphorylated during mitosis and then – like GlsA – associates with the spindle (Matsumoto-Taniura et al., 1996). The remaining four sequence homologs of GlsA and MPP11 include two from mouse, one from C. elegans, and one from Saccharomyces cerevisiae. The presence of GlsA sequence homologs in organisms as divergent as algae, yeast, nematodes and humans suggests that GlsA-related proteins might be found in all eukaryotes. A Asymmetric divisions 60 x Gonidia/embryos x Somatic cells 50 Relative 40 Abundance of glsA Transcript 30 20 10 0 x x x x x 40 44 48/0 4 8 Cleavage period x x x x x 12 16 20 24 28 32 36 40 Hour in life cycle glsA encodes a protein highly similar to several known J proteins, including human MPP11 BLAST analysis (Altschul et al., 1990) of the glsA coding sequence revealed that the polypeptide it encodes, GlsA, includes an approx. 70-aa motif, the J domain, that is found in a large family of proteins, called J proteins, that includes the Hsp40 class of molecular chaperones, as well as many other proteins believed to have chaperone-like functions (Caplan et al., 1993). In all reported test cases, the J domain is indispensable for the function of the J protein in which it occurs. The J domain of Fig. 4. Accumulation of glsA RNA and GlsA protein in time and space. (A) Relative GlsA is identical to the consensus J-domain abundance of glsA RNA in both cell types measured by semi-quantitative RT-PCR. The sequence at all nine amino acid positions that somatic cells present from hour zero (the beginning of the cleavage period) through hour 12 are those of the parental spheroids within which the embryos are developing; at later are known to be highly conserved, including stages 2 generations of somatic cells (differing in age by 48 hours) are present. three that are invariant in all known J (B) Western-blot analysis of GlsA protein in embryos and postembryonic juveniles of proteins (Fig. 5A). Of the many J proteins in strain 22gls1-NRG. (C-E) Indirect immunofluorescence images of a dividing 4-cell embryo the data base, five share extensive regions of of strain 22gls-NRG stained with anti-β-tubulin (C), anti-HA (D) and DAPI (E). Note that similarity with GlsA outside the J domain, in the HA-tagged protein is localized predominantly in the vicinity of the mitotic spindle. 3 regions that we designate WR1, WR2, and Scale bar, 10 µm. 654 S. M. Miller and D. L. Kirk Q A * * A B GlsA *** * GlsA DnaJ DP Y SL L GL A N ER W TA S E A Q I RA A Y R K TC L E N H P DK A L I GlsA DnaJ N VT D E AE R ER I V EH F KT I QD A Y DI L SD P A K R R EF D S T D J M WR1 WR2 MIDA1 DY Y E I L G V S K - - - T A E E R E I R K A Y K R L A M K Y H P DR N Q G MPP11 * * * D KE A E AK - -- - - -- F KE I KE A Y EV L TD S Q K R A AY D Q Y G Zrf1 CeZrf Zuotin Fig. 5. Comparison of GlsA with other J proteins. (A) The J domains of GlsA and E. coli DnaJ. Identical residues are boxed and shaded; similar ones are boxed and unshaded. Large asterisks indicate residues that are fully conserved in all J domains; small asterisks indicate highly conserved residues. Arrows indicate residues that were mutated in plasmid LV399. (B) Domain structure of GlsA and five proteins of highly similar sequence. The function(s) of the WR (tryptophane repeat) domains is unknown; the functions of the J and M domains and the MPP11 and MIDA1 proteins are discussed in the text. The species of origin and the GenBank accession numbers of the other three proteins are: Zrf1, mouse, 1731448; CeZrf, C. elegans, 1572812; Zuotin, S. cerevisiae, 418611. The J domain is indispensable for GlsA function To test whether the J domain plays an essential role in the asymmetric-division function of GlsA, we generated a mutant glsA gene that encodes a defective J domain within an otherwise wild-type protein. The residues that are invariant in all known J domains are a his-pro-asp tripeptide approximately 30 residues from the amino end of the domain (Fig. 5A). Replacement of this histidine with glutamine inactivates two prototypical J proteins: the DnaJ/Hsp40 of E. coli, and its S. cerevisiae homolog, Ydj1 (Sell et al., 1990; Tsai and Douglas, 1996). We introduced this same mutation into glsA (along with a second mutation that converts the aspartate to alanine, in order to generate a novel NgoMI restriction site), and used a plasmid bearing the mutant gene (LV399) to attempt rescue of the Gls phenotype of strain 22gls1 via co-transformation. Plasmid LV377, with the wild-type gene, was used in parallel to transform equal aliquots of a single sample of mutant spheroids. Whereas 38 of 57 Nit+ transformants were converted to Gls+ by the control plasmid, LV377, none of 41 Nit+ transformants were converted to Gls+ by plasmid LV399. DNA blot analysis revealed that 7 of the 10 Nit+ Gls− transformants from the latter group that we tested had stably incorporated the mutant glsA transgene (representative data in Fig. 6A), indicating that the failure to rescue the phenotype was not due to a failure to incorporate the mutant gene. As expected, all Gls+ Nit+ transformants that were tested contained both the endogenous and transgenic copies of the glsA gene (Fig. 6B). We conclude that the J domain of GlsA is indispensable for its function in repositioning the cleavage plane during asymmetric cell divisions. glsA is not the only locus that can mutate to produce a Gls phenotype Gls strains have not been amenable to Mendelian analysis, probably because the cellular composition and arrangement of a Gls spheroid precludes the sequential sperm-somatic cell and sperm-egg interactions that are required for completing fertilization (Coggin et al., 1979; Starr, 1969). As a result, we have been unable to determine by conventional Mendelian methods how many loci are defined by the Gls mutants in our collection. However, the cloned glsA locus permitted us to begin to address this question. First we tested the ability of the glsA gene carried on plasmid LV377 to rescue the Gls phenotype of two independent mutants, 22gls4 and 22gls5, and found it was unable to rescue either of them. Then we used the glsA gene to probe Southern blots containing genomic DNAs from over 40 independent Gls mutants, including 10 that are highly revertible (and hence likely to be transposon induced). Fig. 6. Mutation of the J domain renders GlsA nonfunctional. (A) Southern blot of NgoMI-digested DNAs probed with P2, (see Fig. 3B) demonstrating that plasmid LV399 was incorporated into many Nit+ Gls− transformants. Arrows indicate wild-type (2.6 kb), mutant (4.2 kb), and transgene (0.9 and 1.7 kb) glsA fragments. (B) Southern blot of BamHI-digested DNAs probed with P1, demonstrating that stable incorporation of plasmid LV377 accompanied rescue of Gls phenotype in all five Nit+ Gls+ transformants (T1-T5). T6 is a Nit+ Gls− transformant included as a control. Arrows indicate wild-type (1.8 kb) and mutant (3.4 kb) fragments. The Volvox glsA gene 655 Fig. 7. One possible mechanism of action of GlsA. 2MTR In Volvox and its relatives, the 4MTR 4MTR AA position assumed by the basal BB AA AA AAAAAA body apparatus (BBA) – and 2MTR BB 2MTR 2MTR BB 2MTR AAAAAA 70 more specifically by the 4BB AAAAAA p BB n Hs mai AA AAAAAA membered microtubule rootlets AA AAAAAA DoAAAAAAA ain J 4MTR 4MTR AAAAAAA (4MTRs) that are attached to om D AAAAAAA in e e M BBA location at telophase BBA location at telophase the basal bodies (BB) – by the sit ran AAAAAAA M mb e AAAAAAA preceding a symmetric preceding an asymmetric m end of telophase n defines the GlsA AAAAAAA division of -a wild type or division in a wild type AAAAAAA plane of division n+1 (Kirk, glsA embryo embryo 4MTR 1998). One mechanism of GlsA action would be that while the BBA is moving about during the telophase preceding an asymmetric division, GlsA molecules bound (via their J domains) to Hsp70 molecules on the BBA contact and bind (via their M domains) to eccentrically localized membrane proteins, M, thereby tethering the BBA to an off-center site. Other possible mechanisms are discussed in the text. None of the strains tested had detectable polymorphisms at the glsA locus, consistent with the notion that mutations in a gene or genes other than glsA caused the Gls phenotype of at least some of these mutants. DISCUSSION glsA encodes a J protein that is required for asymmetric cell division We have established that the glsA gene encodes a polypeptide containing the well defined approximately 70-aa motif, the J domain, that is found in a diverse family of proteins, most notably the Hsp40 class of molecular chaperones (Caplan et al., 1993). We have also shown that inactivating this domain by in vitro mutagenesis abolishes the ability of glsA to restore to V. carteri embryos the capacity for asymmetric division that is essential for establishing its two cell types – germ and soma. The fact that the five proteins in the data base that are most similar in sequence and domain structure to the deduced GlsA protein are found in organisms as different from the green alga V. carteri as yeast, nematodes and humans raises the possibility that this recently discovered subfamily of J proteins may be ubiquitous in eukaryotes, and may even have some generalized role in cell division. Should that be so, what is learned about the role of GlsA in Volvox cell division may have considerably broader significance than it might have appeared initially. The prototypical J protein is E. coli DnaJ/Hsp40, which is required, together with DnaK/Hsp70, for replication of phage λ. In that role and many others, including protection of cells from potentially lethal heat shock, DnaJ binds to DnaK, targets it to specific substrates, and stimulates its ATPase activity (Cyr et al., 1994). The J domain is the region required both for binding to DnaK and for stimulating its ATPase activity. Other domains present in DnaJ include a cysteine-rich region (CRR) required for its substrate recognition and protein folding activities, and a ‘hinge’ region. Homologs of DnaJ/Hsp40 and DnaK/Hsp70 have been found in every organism examined, and many of these DnaJ homologs that also possess the CRR and hinge regions are believed to be functional homologs of E. coli Hsp40, involved in mediating the stress response (Cyr et al., 1994). But what of proteins, like GlsA, that share with DnaJ only the J domain? Whereas Hsp70s are all very similar in structure, eukaryotic J proteins are extremely variable, modular proteins: the J domain (which is usually near the N terminus) can be combined with many different C-terminal modules. Studies of the various J proteins and Hsp70 homologs of yeast (Brodsky and Schekman, 1993; Caplan et al., 1992; Schlenstedt et al., 1995; Zhong and Arndt, 1993) have led to the view that small differences in J domain sequence specify which Hsp70 isoforms various J proteins will pair with, and different Cterminal domains act as ‘adapters’ that determine where the Hsp70/J-protein complex will localize in the cell, what substrates it will bind to, and what functions it will execute (Kelley, 1998; Silver and Way, 1993). Over time, the number of roles that various J proteins and Hsp70s have been found to play in the life of the cell has grown continuously. Beyond the role they were initially found to play in thermoprotection (by binding and preventing aggregation of thermally unfolded proteins, promoting proper refolding of some, and targeting others for proteolysis), J protein/Hsp70 complexes are now known to be involved in protein synthesis, targeting and translocation of specific proteins into various organelles, and assembly of a diverse array of multi-protein complexes (Caplan et al., 1993; Craig et al., 1994; Cyr et al., 1994). Moreover, they can interact with other Hsps (such as Hsp60 or Hsp90) to promote yet more activities (Kimura et al., 1995; Lewis et al., 1992). In all such cases, no role has ever been defined for the J domain other than as a site required for binding to and activating an Hsp70 partner. But in order to act as an adapter capable of targeting a specific Hsp70 to a particular substrate or intracellular site, a J protein requires a second binding site. A second domain in GlsA that is a candidate to serve such a function is what we call the ‘M domain’, because it was first identified in MIDA1 (one of two mouse homologs of GlsA) as the binding site by which MIDA1 attaches to the ‘Id’ protein and thereby inhibits the activity of Id as a positive regulator of growth and a negative regulator of differentiation in erythroleukemic cells (Shoji et al., 1995). Our working hypothesis is that the M domain of GlsA serves as a second protein-binding site that is used by GlsA to bind its cognate Hsp70 to a specific cellular target. Therefore, we are presently using biochemical and molecular genetic methods to isolate proteins that bind GlsA, in a search for its postulated cognate Hsp70 and second-target molecules. Meanwhile, we may ask: where in the cell might the protein partners of GlsA be located, and how might they act in concert to shift the division plane? 656 S. M. Miller and D. L. Kirk A possible mechanism of action for GlsA Several considerations lead us to speculate that the GlsAHsp70 interaction occurs right on the cell-division apparatus, and that it plays a relatively direct role in establishing the cell division plane. These considerations include: (1) One of the closest homologues of GlsA in the data base, the human mitotic phase phosphoprotein 11, or MPP11, has been found in association with the mitotic spindle after it has been phosphorylated by a cyclin-dependent kinase (MatsumotoTaniura, 1996). (2) Although we have yet to establish whether GlsA is also phosphorylated during M phase, we have demonstrated here (Fig. 4C-E) that it certainly is associated with the mitotic spindle in symmetrically dividing blastomeres of early Volvox embryos – although it remains to be determined how this localization may be modified in cells preparing to divide asymmetrically. (3) There is growing recognition that Hsp70s have important roles in regulating MT polymerization, centrosome function and mitotic spindle assembly: In Chlamydomonas, the closest unicellular relative of Volvox, an Hsp70 has been found at the tip of the flagellum, at the site where axonemal MTs are assembled (Bloch and Johnson, 1995), and an Hsp70 is part of a complex that is required in yeast for the proper assembly of mitotic spindles (Ursic and Culbertson, 1991). Furthermore, a constitutively expressed Hsp70 localizes to the centrosomes of mitotic animal cells (Brown et al., 1996a), and a stress-induced Hsp70 that localizes to the centrosome following heat shock is required for restoration of structure and microtubule organizing (MTOC) function to a thermally inactivated centrosome (Brown et al., 1996b). It remains to be determined whether J proteins are paired with the Hsp70 species that are now known to be present in such MTOCs, but given the many other Hsp70 functions that are known to require J-protein partners, it is reasonable to assume that this one does also. (4) We have recently established that anti-Hsp70 antibodies stain the mitotic-spindle regions of cleaving Volvox embryos in a pattern similar to that illustrated in Fig. 4 for HA-tagged GlsA (data not shown). All these considerations lead us to speculate that GlsA may function in asymmetric division by complexing with an Hsp70 that is a structural component of the Volvox division apparatus, and that formation of this complex ultimately leads to formation of a cleavage furrow at an off-center location. Visualization of the ways in which a GlsA-Hsp70 complex on the division apparatus might lead to formation of an eccentric furrow will be facilitated by a brief discussion of the nature of the cell division apparatus in the Volvocales (the order of green flagellates to which Volvox belongs), because the volvocalean division apparatus is as different from the division apparati of plants or animals as those are from one another (reviewed in more detail, with fuller documentation, by Kirk, 1998). In all volvocalean cells, the basal bodies (BBs) act as the organizational centers of the cell throughout the cell cycle, being connected directly or indirectly to all other organelles via a complex (yet highly regular) array of cytoskeletal elements that constitute parts of the basal-body apparatus, or BBA, which is the centrosome equivalent of the volvocalean cell. Whereas these BBs sit at the bases of the flagella during interphase, during mitosis the cells are devoid of flagella, yet the BBs remain attached to the plasmalemma – and to an array of other organelles – while serving as centrioles at the poles of the mitotic spindle. With respect to division-plane specification, the most important cytoskeletal attachments to the BBs are a cruciate array of four ‘microtubule rootlets’ (MTRs) that radiate from the BBs beneath the plasmalemma. Rootlets containing two and four microtubules (2MTRs and 4MTRs, respectively) alternate in this cruciate array (Fig. 7). The location of the 2MTRs throughout interphase predicts the axis in which the next mitotic spindle will elongate, whereas the position of the 4MTRs not only predicts, it specifies the plane in which the cell will next divide (Ehler et al., 1995). The reason that these relationships are generalizable is that during prophase volvocalean BBs separate (as the spindle forms and elongates between them) in the direction that is defined by their associated 2MTRs, whereas during telophase the 4MTRs act as the MTOCs for two parallel sheets of ‘cleavage microtubules’ that define the plane in which the cleavage furrow will ingress (Ehler and Dutcher, 1998; Gaffal and el-Gammal, 1990). While cytokinesis is progressing, the (newly duplicated) BBs perform an additional series of directed movements that are of particular importance in the present context: while still attached to the plasmalemma, they move in a complex three-dimensional trajectory back toward and along the developing cleavage furrow, while undergoing a partial rotation, eventually settling down in predictable locations and orientations – whereupon a new cruciate array of MTRs is formed around them. The reason that this complex (and as yet incompletely described) set of telophase movements of the BBA seems particularly important to us is because all present indications are that the locations and orientations that are assumed by the BBs and their new MTRs at the end of this set of movements in division cycle n determine the plane of division n+1. In most volvocalean cell cycles, the new BBA formed at the end of any division cycle is positioned in such a way that the next division will be symmetrical. But our working hypothesis is that it is at this particular stage – at telophase of division n – that GlsA and the proteins with which it associates act to position the BBA eccentrically, thereby preparing the cell to divide asymmetrically during division cycle n+1. We can visualize at least three quite different ways in which a GlsA-Hsp70 complex on the BBA might act, in conjunction with other factors, to prepare a blastomere for asymmetric division: (1) In the simplest version, as the BBA with its associated GlsA-Hsp70 complex is moving about at the end of division cycle n, GlsA makes contact with and binds to its second ligand, ‘M’ (which is located on the plasmalemma at an eccentric site) and this causes the BBA to become tethered to this eccentric site (Fig. 7). (2) Alternatively, GlsA might act as an adapter that links the BBA Hsp70 to a cytoplasmic protein, such as a microtubule motor protein, that is required to move the BBA along the underside of the plasmalemma to its predestined off-center location. (Such a scheme would involve an interesting mechanistic parallel to asymmetric division in early C. elegans embryos, where a microtubule motor appears to be involved in moving the centrosomal apparatus with respect to an eccentric binding site on the membrane after division n, to prepare the cell for division n+1; Waddle et al., 1994). (3) Finally, in parallel with the roles of certain other J-protein-Hsp70 partnerships in protein synthesis and several other cellular functions (Caplan et al., 1993; Craig et al., 1994; Cyr et al., 1994), the postulated GlsA-Hsp70 complex of Volvox may function by directing the assembly of The Volvox glsA gene a multi-protein complex (presumably BBA associated) that will then act, independently of GlsA and Hsp70, to effect eccentric positioning of the BBA. Sorting out which if any of these possibilities is realized in dividing Volvox embryos will require identification and characterization of molecules to which GlsA binds, as well as elucidation of the spatial distribution of GlsA and its binding partners in blastomeres that are preparing to divide asymmetrically. In summary, because asymmetric cell division is so crucial to Volvox cell fate determination, the isolation of the glsA gene is an important step in our attempts to understand how the division of labor between the two cell types of this model organism is programmed and executed. But it is only a first step. The study of additional gls genes, and of factors that interact with GlsA, will be required to provide a more complete picture of how positioning of the cell division plane is controlled in Volvox, and possibly in other multicellular organisms as well. We thank L. Duncan, M. Kirk, G. Köhl, J. McNally, I. Nishii, B. Taillon and C. Wagner for helpful discussions and/or comments on an earlier version of the manuscript, P. Bommarito, M. B. Karr and W. Müller for technical support, L. Ellis for providing the anti-HA monoclonal antibody, J. Rosenbaum for providing the anti-β-tubulin polyclonal antibody and I. Nishii for advice regarding preparation of V. carteri embryos for immunocytology. This work was supported by an NSF postdoctoral fellowship (#BIR-92-03682) to S. M., and by research grants from the NSF (no. MCB-9304447) to D. K., and from the USDA (no. 97-3504-4626) to D. K. and S. M. REFERENCES Altschul, S. F., Gish, W., Miller, W., Myers, E. W. and Lipman, D. J. (1990). Basic local alignment search tool. J. Mol. Biol. 215, 403-410. Atassi, M. Z. and Webster, R. G. (1983). Localization, synthesis, and activity of an antigenic site on influenza hemaglutinin. Proc. Natl. Acad. Sci. USA 80, 840-844. Bloch, M. A. and Johnson, K. A. (1995). Identification of a molecular chaperone in the eukaryotic flagellum and its localization to the site of microtubule assembly. J. Cell Sci. 108, 3541-3545. Broadus, J. and Doe, C. Q. (1997). Extrinsic cues, intrinsic cues, and microfilaments regulate asymmetric protein localization in Drosophila neuroblasts. Curr. Biol. 7, 827-835. Brodsky, J. L. and Schekman, R. (1993). A sec63-BiP complex from yeast required for protein translocation in a reconstituted proteoliposome. J. Cell Biol. 123, 1355-1363. Brown, C. R., Doxsey, S. J., Hong-Brown, L. Q., Martin, R. L. and Welch, W. J. (1996a). Molecular chaperones and the centrosome. A role for TCP1 in microtubule nucleation. J. Biol. Chem. 271, 824-832. Brown, C. R., Hong-Brown, L. Q., Doxsey, S. J. and Welch, W. J. (1996b). Molecular chaperones and the centrosome. A role for hsp 73 in centrosomal repair following heat shock treatment. J. Biol. Chem. 271, 833-840. Caplan, A. J., Cyr, D. M. and Douglas, M. G. (1992). YDJ1p facilitates polypeptide translocation across different intracellular membranes by a conserved mechanism. Cell 71, 1143-1155. Caplan, A. J., Cyr, D. M. and Douglas, M. G. (1993). Eukaryotic homologues of Escherichia coli DnaJ: a diverse protein family that functions with HSP70 proteins. Mol. Biol. Cell 4, 555-563. Coggin, S. J., Hutt, W. and Kochert, G. (1979). Sperm bundle-female somatic cell interaction in the fertilization process of Volvox carteri f. weismannia. J. Phycol. 15, 247-251. Craig, E. A., Baxter, B. K., Becker, J., Halladay, J. and Ziegelhoffer, T. (1994). Cytosolic hsp70s of Saccharomyces cerevisiae: roles in protein synthesis, protein translocation, proteolysis, and regulation. In The Biology of Heat Shock Proteins and Molecular Chaperones (ed. R. I. Morimoto, A. Tissiéres and N. Georgopoulos), pp. 31-52. Plainview, NY: Cold Spring Harbor Laboratory Press. 657 Cyr, D. M., Langer, T. and Douglas, M. G. (1994). DnaJ-like proteins: molecular chaperones and specific regulators of Hsp70. Trends Biochem. Sci. 19, 176-181. Di Laurenzio, L., Wusocka-Diller, J., Malamy, J. E., Pysh, L., Helariutta, Y., Freshour, G., Hahn, M. G., Feldman, K. A. and Benfey, P. N. (1996). The SCARECROW gene regulates an asymmetric cell division that is essential for generating the radial organization of the Arabidopsis root. Cell 86, 423-433. Doe, C. Q. (1996). Spindle orientation and asymmetric localization in Drosophila: Both Inscuteable? Cell 86, 695-697. Ehler, L. L. and Dutcher, S. K. (1998). Pharmacological and genetic evidence for a role of rootlet and phycoplast microtubules in the positioning and assembly of cleavage furrows in Chlamydomonas reinhardtii. Cell Motil. Cytoskeleton 40, 193-207. Ehler, L. L., Holmes, J. A. and Dutcher, S. K. (1995). Loss of spatial control of the mitotic spindle apparatus in a Chlamydomonas reinhardtii mutant strain lacking basal bodies. Genetics 141, 945-960. Field, J., Nikawa, J.-J., Broek, D., MacDonald, B., Rodgers, L., Wilson, I. A., Lerner, R. and Wigler, M. (1988). Purification of a RAS-responsive adenyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method. Mol. Cell. Biol. 8, 2159-2165. Gaffal, K. P. and el-Gammal S. (1990). Elucidation of the enigma of the “metaphase band” of Chlamydomonas reinhardtii. Protoplasma 156, 139148. Goodner, B. and Quatrano, R. S. (1993). Fucus embryogenesis: a model to study the establishment of polarity. Plant Cell 5, 1471-1481. Guo, S. and Kemphues, K. J. (1996). A non-muscle myosin required for embryonic polarity in Caenorhabditis elegans. Nature 382, 455-458. Horvitz, H. R. and Herskowitz, I. (1992). Mechanisms of asymmetric cell division: two Bs or not two Bs, that is the question. Cell 68, 237-255. Kelley, W. L. (1998). The J-domain family and the recruitment of chaperone power. Trends Biocem. Sci. 23, 222-227. Kimura, Y., Yahara, I. and Lindquist, S. (1995). Role of the protein chaperone YDJ1 in establishing Hsp980-mediated signal transduction pathways. Science 268, 1362-1365. Kirk, D. L. (1998). Volvox: The Molecular Genetic Origins of Multicellularity and Cellular Differentiation. Cambridge: Cambridge University Press. Kirk, D. L., Kaufman, M. R., Keeling, R. M. and Stamer, K. A. (1991). Genetic and cytological control of the asymmetric divisions that pattern the Volvox embryo. Development Supplement 1, 67-82. Kirk, M. M. and Kirk, D. L. (1985). Translational regulation of protein synthesis, in response to light, at a critical stage of Volvox development. Cell 41, 419-428. Kirk, M. M., Ransick, A., McRae, S. E. and Kirk, D. L. (1993). The relationship between cell size and cell fate in Volvox carteri. J. Cell Biol. 123, 191-208. Kraut, R., Chia, W., Jan, L. Y., Jan, Y. N. and Knoblich, J. A. (1996). Role of inscuteable in orienting asymmetric cell divisions in Drosophila. Nature 383, 50-55. Lewis, V. A., Hynes, G. M., Zheng, D., Saibil, H. and Willison, K. (1992). T-complex polypeptide-1 is a subunit of a heteromeric particle in the eukaryotic cytosol. Nature 358, 249-252. Lloyd, C. W. (1991). The Cytoskeletal Basis of Plant Growth and Form. New York: Academic Press. Mages, W., Salbaum, J. M., Harper, J. F. and Schmitt, R. (1988). Organization and structure of Volvox α-tubulin genes. Mol. Gen. Genet. 213, 449-458. Matsumoto-Taniura, N., Pirollet, F., Monroe, R., Gerace, L. and Westendorf, J. M. (1996). Identification of novel M phase phosphoproteins by expression cloning. Mol. Biol. Cell 7, 1455-1469. McGrail, M. and Hays, T. S. (1997). The microtubule motor cytoplasmic dynein is required for spindle orientation during germline cell divisions and oocyte differentiation in Drosophila. Development 124, 2409-2419. Miller, S. M., Schmitt, R. and Kirk, D. L. (1993). Jordan, an active Volvox transposable element similar to higher plant transposons. Plant Cell 5, 11251138. Raff, J. W. and Glover, D. M. (1989). Centrosomes, not nuclei, initiate pole cell formation in Drosophila embryos. Cell 57, 611-619. Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. Schiedlmeier, B., Schmitt, R., Müller, W., Kirk, M. M., Gruber, H., Mages, W. and Kirk, D. L. (1994). Nuclear transformation of Volvox carteri. Proc. Natl. Acad. Sci. USA 91, 5080-5084. 658 S. M. Miller and D. L. Kirk Schlenstedt, G. S., Harris, S., Risse, B., Lill, R. and Silver, P. A. (1995). A yeast DnaJ homologue, Scj1p, can function in the endoplasmic reticulum with BiP/Kar2p via a conserved domain that specifies interaction with Hsp70s. J. Cell Biol. 129, 979-988. Schmitt, R., Fabry, S. and Kirk. D. L. (1992). In search of the molecular origins of cellular differentiation in Volvox and its relatives. Int. Rev. Cytol. 139, 189-265. Sell, S. M., Eisen, C., Ang, D., Zylicz, M. and Georgopoulos, C. (1990). Isolation and characterization of dnaJ mutants of Escherichia coli. J. Bacteriol. 172, 4827-4835. Shoji, W., Inoue, T., Yamamoto, T. and Obinata, M. (1995). MIDA1, a protein associated with Id, regulates cell growth. J. Biol. Chem. 270, 2481824825. Silver, P. A. and Way, J. C. (1993). Eukaryotic DnaJ homologs and the specificity of Hsp70 activity. Cell 74, 5-6. Skop, A. R. and White, J. G. (1998). The dynactin complex is required for cleavage plane specification in early Caenorhabditis elegans embryos. Curr. Biol. 8, 1110-1116. Starr, R. C. (1969). Structure, reproduction, and differentiation in Volvox carteri f. nagariensis Iyengar, strains HK 9 and 10. Arch. Protistenkd. 111, 204-222. Starr, R. C. (1970). Control of differentiation in Volvox. Dev. Biol. (suppl.) 4, 59-100. Strome, S. (1993). Determination of Cleavage Planes. Cell 72, 3-6. Stukenberg, P. T., Lustig, K. D., McGarry, T. J., King, R. W., Kiang, K. and Kirschner, M. W. (1997). Systematic identification of mitotic phosphoproteins. Curr. Biol. 7, 338-348. Tam, L.-W. and Kirk, D. L. (1991). The program for cellular differentiation in Volvox carteri as revealed by molecular analysis of development in a gonidialess/somatic regenerator mutant. Development 112, 571-580. Tsai, J. and Douglas, M. G. (1996). A conserved HPD sequence of the Jdomain is necessary for YDJ1 stimulation of Hsp70 ATPase activity at a site distinct from substrate binding. J. Biol. Chem. 271, 9347-9354. Ursic, D. and Culbertson, M. (1991). The yeast homologue to mouse TCP1 affects microtubule mediated processes. Mol. Cell. Biol. 11, 2629-2640. Waddle, J. A., Cooper, J. A. and Waterston, R. H. (1994). Transient localized accumulation of actin in Caenorhabditis elegans blastomeres with oriented asymmetric divisions. Development 120, 2317-2328. White, J. and Strome, S. (1996). Cleavage plane specification in C. elegans: How to divide the spoils. Cell 84, 195-198. Zhong, T. and Arndt, K. (1993). The yeast S1S1 protein, a DnaJ homolog, is required for the initiation of translation. Cell 73, 1175-1186. Zwaal, R. R., Ahringer, J., van Luenen, H. G. A. M., Rushforth, A., Anderson, P. and Plasterk, R. H. A. (1996). G proteins are required for spatial orientation of early cell cleavages in C. elegans embryos. Cell 86, 619-629.