candida albicans kinesin kar3 depends on a cik1-like ... · kar3-green ﬂuorescent protein...

Candida albicans Kinesin Kar3 Depends on a Cik1-Like RegulatoryPartner Protein for Its Roles in Mating, Cell Morphogenesis, andBipolar Spindle Formation

Corey Frazer,a Monika Joshi,a Caroline Delorme,a Darlene Davis,a Richard J. Bennett,b John S. Allinghama

Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, Ontario, Canadaa; Department of Molecular Microbiology and Immunology, BrownUniversity, Providence, Rhode Island, USAb

Candida albicans is a major fungal pathogen whose virulence is associated with its ability to transition from a budding yeastform to invasive hyphal filaments. The kinesin-14 family member CaKar3 is required for transition between these morphologi-cal states, as well as for mitotic progression and karyogamy. While kinesin-14 proteins are ubiquitous, CaKar3 homologs inhemiascomycete fungi are unique because they form heterodimers with noncatalytic kinesin-like proteins. Thus, CaKar3-basedmotors may represent a novel antifungal drug target. We have identified and examined the roles of a kinesin-like regulator ofCaKar3. We show that orf19.306 (dubbed CaCIK1) encodes a protein that forms a heterodimer with CaKar3, localizes CaKar3 tospindle pole bodies, and can bind microtubules and influence CaKar3 mechanochemistry despite lacking an ATPase activity ofits own. Similar to CaKar3 depletion, loss of CaCik1 results in cell cycle arrest, filamentation defects, and an inability to undergokaryogamy. Furthermore, an examination of the spindle structure in cells lacking either of these proteins shows that a large pro-portion have a monopolar spindle or two dissociated half-spindles, a phenotype unique to the C. albicans kinesin-14 homolog.These findings provide new insights into mitotic spindle structure and kinesin motor function in C. albicans and identify a po-tentially vulnerable target for antifungal drug development.

Candida albicans is a common commensal fungus that typicallycauses no harm to the host (1). However, in immunocompro-

mised individuals, neonates, and patients under intensive care, itcan cause serious skin and mucosal infections as well as life-threat-ening systemic infections (1, 2). It is also able to form tenaciousbiofilms on implanted medical devices (3). A major factor facili-tating C. albicans virulence is its ability to readily switch betweengrowth as a yeast, pseudohyphae, and hyphae (1, 4). In the yeastmode, daughter cells bud off and dissociate from the mother cell,while pseudohyphal cells remain connected at constricted septa-tion sites (4). Hyphae are the invasive form and are important forvirulence during systemic infections as well as for biofilm forma-tion (1, 5, 6). In all of these morphological states, microtubule-associated motor proteins play major roles in microtubulecytoskeleton remodeling, nuclear movements, chromosome seg-regation, and cargo transport (7–9). This provides a rationale fortheir use as novel targets for antifungal drugs (10).

C. albicans Kar3 (CaKar3) is a kinesin-14 family member thathas been shown to function in mitotic division and is critical forhypha formation and nuclear fusion during mating (9, 11). Itshomolog in Saccharomyces cerevisiae has similar functions in mi-tosis and mating that are enabled by interactions with two kinesin-like proteins: S. cerevisiae Cik1 (ScCik1) and ScVik1 (12–20).ScKar3 forms complexes with each of these proteins via a centralcoiled-coil domain to form parallel heterodimers that are struc-turally similar to many other dimeric kinesins yet are functionallyvery different because ScCik1 and ScVik1 lack residues requiredfor ATP binding (16, 19, 20). Despite this limitation, ScCik1 andScVik1 can each bind microtubules (20, 21), and recent studiesshowed that ScKar3/Cik1 and ScKar3/Vik1 complexes move pro-cessively and quite rapidly along microtubules when purified fromyeast lysates (22).

Early studies of budding yeast showed that both ScCik1 and

ScKar3 localize to the spindle pole body (SPB) in vegetativelygrowing cells (15, 18). ScCik1 has also been shown to target ScKar3to interpolar microtubule (ipMT) plus ends of the central spindle(23), where ScKar3/Cik1 complexes are thought to cross-link andstabilize overlapping antiparallel microtubules (12, 13, 18, 24, 25).Recent studies suggest that this facilitates the action of the kine-sin-5 family members ScCin8 and ScKip1 (24, 26), which exertoutwardly directed microtubule sliding forces needed to establishproper metaphase spindle length and for anaphase spindle elon-gation (15, 25, 27–29). ScVik1, on the other hand, targets ScKar3to the spindle poles and has been suggested to directly oppose theforce exerted by ScCin8 and ScKip1 (15, 30, 31). However, itsmode of action is less well studied.

In response to mating pheromones, a truncated ScCik1 iso-form lacking a nuclear localization signal is expressed, while Sc-Vik1 expression is repressed (15, 32). ScKar3 expression also in-creases at this time, and ScCik1 targets ScKar3 to the cytoplasmicmicrotubule plus ends (23) and to the cytoplasmic face of the SPB(12, 15–18). At this point, ScKar3/Cik1 is involved in two stages of

Received 28 January 2015 Accepted 26 May 2015

Accepted manuscript posted online 29 May 2015

Citation Frazer C, Joshi M, Delorme C, Davis D, Bennett RJ, Allingham JS. 2015.Candida albicans kinesin Kar3 depends on a Cik1-like regulatory partner proteinfor its roles in mating, cell morphogenesis, and bipolar spindle formation.Eukaryot Cell 14:755–774. doi:10.1128/EC.00015-15.

Address correspondence to John S. Allingham, [email protected].

Supplemental material for this article may be found at http://dx.doi.org/10.1128/EC.00015-15.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

doi:10.1128/EC.00015-15

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the mating process. First, nuclei are moved toward the matingprojection tip, in an ScKar3/Cik1-dependent manner, via cou-pling of ScKar3/Cik1’s minus-end-directed force generation to itsmicrotubule depolymerization activity (33). Second, following fu-sion of polarized cells of opposite mating types, SPB-localizedScKar3/Cik1 is able to shorten interdigitating microtubules ema-nating from the SPBs of opposite nuclei, drawing the two togetherto complete karyogamy (34–36).

We now report the identification and characterization of a ho-mologous Kar3-regulatory protein in C. albicans. Our study dem-onstrates roles for this protein in mitosis, mating, and morpho-genesis of C. albicans. Based on these phenotypes and the sequencefeatures it shares with the S. cerevisiae Kar3-regulatory proteinScCik1, we have dubbed this protein CaCik1. We show that un-derlying the cell cycle arrest phenotype of both CaCIK1 and Ca-KAR3 deletions is a dramatic bipolar spindle formation defect,which is unprecedented for Kar3 and Cik1 homologs in buddingyeast. Therefore, the study of S. cerevisiae motors appears to beinsufficient to explain how spindle assembly is accomplished in C.albicans. We discuss how this could be due to differences in theirkinesin complements following whole-genome duplication(WGD) and the partial loss of duplicated genes in the S. cerevisiaelineage.

MATERIALS AND METHODSCell growth and manipulation. Selection for auxotrophic markers wasconducted using synthetic dropout (SD) medium containing 0.66% yeastnitrogen base (U.S. Biologicals), 0.2% yeast dropout mix lacking uracil,arginine, leucine, and histidine (U.S. Biologicals), 2% dextrose (Bioshop),and 200 mg liter�1 uridine and supplemented with 200 mg liter�1 histi-dine, leucine, and/or arginine where required. Experimental cultures weregrown to mid-logarithmic phase in completely supplemented dropoutmedium (SDC) unless otherwise indicated. Strains were maintained onyeast extract-peptone-dextrose (YPD) plates, containing 1% yeast extract(BD-Bacto), 2% peptone (BD-Bacto), and 2% dextrose (Bioshop). YPDwas supplemented with 100 �g ml�1 nourseothricin (CloneNat; WernerBioAgents) for selection of the SAT1 gene.

Gene knockout and fluorescent fusion construction. Gene disrup-tion of the C. albicans CIK1 open reading frame (ORF) (Candida GenomeDatabase tag orf19.306; NCBI gene ID 3638398) was conducted by trans-formation and integration of a linear cassette containing a selectablemarker. A list of C. albicans strains used in this study is presented in Table1. For a list of oligonucleotides used in C. albicans strain construction,please refer to Table S1 in the supplemental material. PCR amplificationwas used to generate disruption cassettes in which a selectable marker wasflanked by approximately 50 bp of C. albicans genomic sequence imme-diately 5= and 3= of the CIK1� coding region. Disruptions were conductedin both the MTLa/MTLa and MTL�/MTL� mating type loci, in parallel.Disruption of CIK1� in a wild-type background (CF026 or CF027) wasconducted sequentially. First, a cik1::LEU2� cassette was amplified frompSN40 (37) by using primers P21 and P22 and transformed into strainsCF026 and CF027 to create strains CF002 and CF005, respectively. Cor-rect cik1::LEU2� cassette integration was confirmed using primers P1/P13and P20/P14 for the upstream and downstream junctions, respectively.Primers P1 and P20 recognize sequences approximately 500 bp upstreamand downstream of the CIK1 ORF. Second, a cik1::HIS1� cassette wasamplified from pSN52 (37) by using the primer pair P21/P22 and thentransformed into strains CF002 and CF005 to create CF013 and CF016,respectively. Integration of the disruption cassette at the correct locationwas confirmed by PCR amplification across the junctions of integration.The cik1::HIS1� sequence was confirmed using primers P1/P12 and P20/P11 for the upstream and downstream junctions, respectively. CIK1� wasdisrupted in a kar3::LEU2�/kar3::HIS� background (CF024, CF025) by

using the ARG4� marker, amplified from pSN69 (37) by using primersP21 and P22, and the SAT1 ClonNAT resistance gene was amplified frompSFS2A by using primers P23 and P24 (38). Correct integration of cik1::ARG4� was confirmed using primer pairs P1/P16 and P20/P15 for theupstream and downstream junctions, respectively. Correct cik1::SAT1 in-tegration was confirmed with primer pairs P1/P17 and P20/P18. In theMTLa/MTLa background (CF025), the first allele of CIK1 was disruptedusing the SAT1 resistance marker to create CF048, and the second allelewas disrupted using ARG4� to create CF061. In the MTL�/MTL� back-ground (CF024), the first allele was disrupted with ARG4� to createCF012, and the second allele was disrupted with SAT1 to create CF019.The absence of additional endogenous CIK1� sequences arising from du-plication or translocation was confirmed in each of the above strains byusing primer pairs P1/P27 and P20/P28.

Fluorescent fusions of CIK1� in a wild-type background were con-structed using the method described by Gerami-Nejad et al. (39) andusing long-tailed primers P32 and P64, with the plasmid pYFP-HIS1(pMG1656) as the template, to create an integration cassette flanked byapproximately 50 bp of the CIK1� ORF immediately before the stopcodon and 50 bp of sequence 3= to the ORF. This cassette was transformedinto the wild type (CF027) to create strain CF069 (CIK1-YFP-HIS1�/CIK1�). Correct integration was confirmed by PCR. The upstream junc-tion was amplified using P28 and P68 (a reverse primer binding in theadh1 terminator), and the downstream junction was amplified using P20and P70 (a forward primer binding in the C. albicans HIS1� gene se-quence of pYFP-HIS1).

To create a Cik1-yellow fluorescent protein (YFP) fusion in the kar3::HIS1�/kar3::LEU2� background, pYFP-HIS1 was modified to containthe SAT1 selectable marker (pYFP-SAT1). pYFP-SAT1 was constructedby excising the HIS1� sequence from pYFP-HIS1 (39) by using EcoRI andtreating the resulting backbone with calf intestinal phosphatase (CIP).The SAT1 sequence was amplified from pSFS2A by using primers P52 andP53, which introduce EcoRI sites. This insert was EcoRI digested andligated into the open, CIP-treated vector backbone. The orientation of theSAT1 sequence in pYFP-SAT1 was determined by restriction fragmentanalysis. A knock-in cassette was then produced by PCR using primersP32 and P33 and transformed into strain CF024 to create CF068 (CIK1-YFP-SAT1/CIK1 kar3::LEU2�/kar3::HIS1�). Correct integration wasconfirmed by amplification across the left flank of the integrated cassette,using primers P28 and P68.

Kar3-green fluorescent protein (Kar3-GFP)-, �-tubulin–red fluores-cent protein (Tub2-RFP)-, and Tub2-GFP-expressing strains were con-structed by generating mobile cassettes from the genomic DNA of CF028(KAR3-GFP-SAT1/KAR3� TUB2-RFP-ARG4�/TUB2�) or CF030 (TUB2-GFP-SAT1/TUB2� HTB1-RFP-ARG4�/HTB1�). For each locus, the inte-grated C-terminal “XFP-adhterm-marker” fragment, along with 200 to250 bp of the ORF and the 3= sequence, was amplified using Phusionpolymerase to generate a cassette for transformation. This simplified thecreation of multiply tagged strains by using small oligonucleotides to cre-ate cassettes with large stretches of homology on either side. As C. albicansis diploid, a smaller PCR product was also generated from the untaggedTub2 locus. The integration cassette was isolated from the PCR mixtureby gel excision and either used directly to transform cells or given an A tailby incubation with Taq polymerase and dATP and cloned into pGEM-TEasy (Promega), which was then used as a template for cassette amplifi-cation. A KAR3-GFP-SAT1 cassette was generated using primers P56 andP57. Integration of this cassette was confirmed by PCR across the entirelocus by using primers P58 and P59, by which heterozygous KAR3-GFP-SAT1/KAR3� strains could be differentiated from untransformed cells bythe presence of a large insertion in one of the KAR3 loci. TUB2-RFP-ARG4� and TUB2-GFP-SAT1 cassettes were generated similarly, usingprimers P60 and P61. Primers P62 and P63 were used to confirm thecorrect insertion. Kar3-GFP was transformed into wild-type (CF027) andcik1�/� (CF016) strains to create CF051 (KAR3-GFP-SAT1/KAR3�) andCF056 (KAR3-GFP-SAT1/KAR3� cik1::LEU2�/cik1::HIS1�). To create

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TABLE 1 Names, genotypes, mating types, and sources of the strains used in this study

Strain Genotype (brief) Genotype (full)aMatingtype Parent (source)

CF026 Wild type his1�/� leu2�/� arg4�/� a/a RBY1132b

CF027 Wild type his1�/� leu2�/� arg4�/� �/� RBY1133b

CF119 Wild type � vector RPS1�/rps1::pCIp10-SAT1 his1�/� leu2�/� arg4�/� �/� CF027 (this study)CF025 kar3�/� kar3::LEU2�/kar3::HIS1� his1�/� leu2�/� arg4�/� a/a RSY12b

CF024 kar3�/� kar3::LEU2�/kar3::HIS1� his1�/� leu2�/� arg4�/� �/� RSY11b

CF123 kar3�/� � vector kar3::LEU2�/kar3::HIS1� RPS1�/rps1::pCIp10-SAT1 his1�/� leu2�/� arg4�/� �/� CF024 (this study)CF125 kar3�/� � KAR3� kar3::LEU2�/kar3::HIS1� RPS1�/rps1::pCIp10-SAT1-KAR3� (�1,000 bp)

his1�/� leu2�/� arg4�/��/� CF024 (this study)

CF002 cik1�/CIK1� CIK1�/cik1::LEU2� his1�/� leu2�/� arg4�/� a/a CF026 (this study)CF013 cik1�/� cik1::LEU2�/cik1::HIS1� his1�/� leu2�/� arg4�/� a/a CF002 (this study)CF005 cik1�/CIK1� CIK1�/cik1::LEU2� his1�/� leu2�/� arg4�/� �/� CF027 (this study)CF016 cik1�/� cik1::LEU2�/cik1::HIS1� leu2�/� his1�/� arg4�/� �/� CF005 (this study)CF126 cik1�/� � vector cik1::HIS1�/cik1::LEU2� RPS1�/rps1::pCIp10-SAT1 his1�/� leu2�/� arg4�/� �/� CF016 (this study)CF129 cik1�/� � CIK1� cik1::HIS1�/cik1::LEU2� RPS1�/rps1::pCIp10-SAT1-CIK1 (�1,000 bp) his1�/�

leu2�/� arg4�/��/� CF016 (this study)

CF048 kar3�/� cik1�/CIK1� CIK1�/cik1::SAT1 kar3::LEU2�/kar3::HIS1� his1�/� leu2�/� arg4�/� a/a CF025 (this study)CF061 kar3�/� cik1�/� kar3::HIS1�/kar3::LEU2� cik1::SAT1/cik1::ARG4� his1�/� leu2�/� arg4�/� a/a CF048 (this study)CF012 kar3�/� cik1�CIK1� CIK1�/cik1::ARG4� kar3::HIS1�/kar3::LEU2� his1�/� leu2�/� arg4�/� �/� CF024 (this study)CF019 kar3�/� cik1�/� kar3::HIS1�/kar3::LEU2� cik1::ARG4�/cik1::SAT1 his1�/� leu2�/� arg4�/� �/� CF012 (this study)CF028 Kar3-GFP Tub2-RFP KAR3�/KAR-GFP-SAT1 TUB2�/TUB2-RFP-ARG4� arg4�/� a/a RSY120b

CF030 Tub2-GFP HTB1-RFP TUB2-GFP-SAT1/TUB2� HTB1-RFP-ARG4�/HTB1� arg4�/� a/a RSY150b

CF057 Tub2-RFP TUB2-RFP-ARG4�/TUB2� his1�/� leu2�/� arg4�/� �/� CF027 (this study)CF132 Tub2-RFP � vector TUB2�/TUB2-RFP-ARG4� RPS1�/rps1::pCIp10-SAT1 his1�/� leu2�/� arg4�/� �/� CF057 (this study)CF058 Tub2-RFP kar3�/� TUB2-RFP-ARG4�/TUB2� kar3::HIS1�/kar3::LEU2� his1�/� leu2�/� arg4�/� �/� CF024 (this study)CF135 Tub2-RFP kar3�/� � vector TUB2�/TUB2-RFP-ARG4� kar3::HIS1�/kar3::LEU2� RPS1�/rps1::pCIp10-

SAT1 his1�/� leu2�/� arg4�/��/� CF058 (this study)

CF136 Tub2-RFP kar3�/� � KAR3� TUB2�/TUB2-RFP-ARG4� kar3::HIS1�/kar3::LEU2� RPS1�/rps1-pCIp10-SAT1-KAR3� (�1,000 bp) his1�/� leu2�/� arg4�/�

�/� CF058 (this study)

CF060 Tub2-RFP cik1�/� TUB2-RFP-ARG4�/TUB2� cik1::LEU2�/cik1::HIS1 his1�/� leu2�/� arg4�/� �/� CF016 (this study)CF139 Tub2-RFP cik1�/� � vector TUB2�/TUB2-RFP-ARG4� cik1::LEU2�/cik1::HIS1� RPS1�/rps1::pCIp10-SAT1


CF141 Tub2-RFP cik1�/� � CIK1� TUB2�/TUB2-RFP-ARG4� cik1::LEU2�/cik1::HIS1� RPS1�/rps1::pCIp10-SAT1-CIK1 (�1,000 bp) his1�/� leu2�/� arg4�/�


CF051 Kar3-GFP KAR3-GFP-SAT1/KAR3� his1�/� leu2�/� arg4�/� �/� CF027 (this study)CF069 Cik1-YFP CIK1-YFP-HIS1�/CIK1� his1�/� leu2�/� arg4�/� �/� CF027 (this study)CF056 Kar3-GFP cik1�/� KAR3-GFP-SAT1/KAR3� cik1::HIS1�/cik1::LEU2� his1�/� leu2�/� arg4�/� �/� CF016 (this study)CF144 Kar3-GFP cik1�/� � vector KAR3�/KAR3-GFP-SAT1 cik1::HIS1�/cik1::LEU2� RPS1�/rps1::pCIp10-ARG4�


CF147 Kar3-GFP cik1�/� � CIK1� KAR3�/KAR3-GFP-SAT1 cik1::HIS1�/cik1::LEU2� RPS1�/rps1::pCIp10-ARG4�-CIK1� (�1,000 bp) his1�/� leu2�/� arg4�/�


CF068 Cik1-YFP kar3�/� CIK1-YFP-SAT1/CIK1� kar3::HIS1�/kar3::LEU2� his1�/� leu2�/� arg4�/� �/� CF024 (this study)CF150 Cik1-YFP kar3�/� � vector kar3::HIS1�/kar3::LEU2� CIK1�/CIK1-YFP-SAT1 RPS1�/rps1-pCIp10-ARG4�


CF153 Cik1-YFP kar3�/� � KAR3� kar3::HIS1�/kar3::LEU2� CIK1�/CIK1-YFP-SAT1 RPS1�/rps1-pCIp10-ARG4�-KAR3� (�1,000 bp) his1�/� leu2�/� arg4�/�


CF075 Tub2-RFP Cik1-YFP TUB2-RFP-ARG4�/TUB2� CIK1-YFP-HIS1�/CIK1� his1�/� leu2�/� arg4�/� �/� CF057 (this study)CF100 Tub2-RFP Kar3-GFP TUB2-RFP-ARG4�/TUB2� KAR3-GFP-SAT1/KAR3� his1�/� leu2�/� arg4�/� �/� CF057 (this study)CF160 Tub2-RFP Spc98-GFP TUB2-RFP-ARG4�/TUB2� SPC98-GFP-SAT1/SPC98� his1�/� leu2�/� arg4�/� �/� CF057 (this study)CF163 Tub2-RFP Spc98-GFP cik1�/� TUB2-RFP-ARG4�/TUB2� SPC98-GFP-SAT1/SPC98� cik1::LEU2�/cik1::HIS1


CF157 Tub2-GFP TUB2�/Tub2-GFP-SAT1 his1�/� leu2�/� arg4�/� �/� CF027 (this study)CF174 Tub2-GFP cik1�/� cik1::HIS1�/cik1::LEU2� TUB2�/Tub2-GFP-SAT1 his1�/� leu2�/� arg4�/� �/� CF016 (this study)CF045 Wild type; opaque his1�/� leu2�/� arg4�/�; opaque a/a CF026 (this study)CF046 Wild type; opaque his1�/� leu2�/� arg4�/�; opaque �/� CF027 (this study)CF034 kar3�/�; opaque kar3::LEU2�/kar3::HIS1� his1�/his1� leu2�/leu2� arg4�/arg4�; opaque a/a CF025 (this study)CF032 kar3�/�; opaque kar3::LEU2�/kar3::HIS1� his1�/his1� leu2�/leu2� arg4�/arg4�; opaque �/� CF024 (this study)CF036 cik1�/�; opaque cik1::LEU2�/cik1::HIS1� his1�/his1� leu2�/leu2� arg4�/arg4�; opaque a/a CF013 (this study)CF042 cik1�/�; opaque cik1::LEU2�/cik1::HIS1� his1�/his1� leu2�/leu2� arg4�/arg4�; opaque �/� CF016 (this study)CF065 kar3�/� cik1�/�; opaque kar3::LEU2�/kar3::HIS1� cik1::SAT1/cik1::ARG4� his1�/his1� leu2�/leu2�

arg4�/arg4�; opaquea/a CF061 (this study)

CF039 kar3�/� cik1�/�; opaque kar3::HIS1�/kar3::LEU2� cik1::ARG4�/cik1::SAT1 his1�/his1� leu2�/leu2�

arg4�/arg4�; opaque�/� CF019 (this study)

a Strains are in the white phase unless otherwise noted. All strains were derived from SN152 (37). The full genotype at the auxotrophic markers is as follows: his1::hisG/his1::hisGleu2::hisG/leu2::hisG arg4::hisG/arg4::hisG ura3::imm434::URA3/ura3::imm434 iro1::IRO1/iro1::imm434.b Described by Sherwood and Bennett (9).

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strains expressing Tub2-RFP in the wild-type, kar3�/�, and cik1�/�backgrounds, the TUB2-RFP-ARG4� cassette was transformed intoCF027, CF024, and CF016, respectively, to create CF057, CF058, andCF060. Strains expressing Tub2-RFP and either Cik1-YFP or Kar3-GFPwere created by transforming a Tub2-RFP-expressing strain (CF057) witheither the KAR3-GFP-SAT1 cassette to yield CF100 or the CIK1-YFP-HIScassette to yield CF075. Strains expressing Tub2-GFP were created bytransforming CF027 and CF016 with the TUB2-GFP-SAT1 cassette tocreate CF157 and CF174, respectively.

Strains expressing Spc98-GFP were constructed using an integrationcassette amplified from pGFP-SAT1, in which the SAT1 selectable markerwas used to replace the HIS1 marker of pGFP-HIS1 (pMG1646), in aprocess identical to the construction of pYFP-SAT1 described above.Primers P108 and P109 were used to amplify the cassette, incorporating ahomologous sequence from the 3= end of orf19.2600, which encodes aputative homolog of S. cerevisiae Spc98 (NCBI gene ID 3640113). Thiscassette was transformed into CF057 to create the Tub2-RFP Spc98-GFPstrain (CF160) and into CF060 to create the Tub2-RFP Spc98-GFPcik1�/� strain (CF163). Correct integration was confirmed by PCR am-plification across the upstream junction of integration, using primersP110 and P18.

To demonstrate that mutant phenotypes are solely a result of loss ofKAR3 and/or CIK1, add-back experiments were conducted to reintroducea wild-type copy of each gene. To this end, pCIp10-based integrationplasmids bearing the SAT1 and ARG4� selectable markers were con-structed (40). To generate pCIp10-SAT1, the NotI/BamHI fragment ofpCIp10, containing the URA3� selectable marker, was replaced by theSAT1 gene from pSFS2A, which was amplified using primers P86 and P87.Similarly, the ARG4� gene, amplified from pSN69 by using primers P97and P98, was used to replace URA3� to create pCIp10-ARG4. Into thesevectors were introduced an MluI/KpnI fragment containing either theKAR3 or CIK1 open reading frame and 1,000 bp of upstream and down-stream sequences; primer pair P90/P91 was used to amplify KAR3�, andprimer pair P95/P96 was used to amplify CIK1�. Integration plasmidswere digested at a StuI site in the RPS1 open reading frame prior to trans-formation. Integration vectors containing the ARG4� marker were usedto create “add-back” and “vector only” strains in the CIK1-YFP kar3�/�and KAR3-GFP cik1�/� backgrounds. Integration vectors containing theSAT1 marker were used to create “add-back” and “vector only” strains inthe wild type and the kar3�/� and cik1�/� disruption strains and thesame strains expressing Tub2-RFP (see Table 1 for details). Confirmationof integration of the pCIp10-SAT1 vectors at the RPS1� locus was con-ducted using PCR primers P92 and P18, and that of the pCIp10-ARG4vectors was done using primers P92 and P16.

Disruption cassettes and StuI-digested complementation plasmidswere transformed into C. albicans by using the lithium acetate-polyethyl-ene glycol (PEG)-heat shock method as previously described (41), withminor modifications. Incubation of cells with the transforming DNA in alithium acetate-PEG solution was carried out for 2 h at 30°C with rotation.Heat shock was conducted at 43°C for 30 min. Transformations involvingselection using the SAT1 gene were accompanied by 2 h of incubation inYPD at 30°C to allow expression of the ClonNAT resistance gene beforeplating.

Karyogamy and nuclear fusion. Opaque-phase cells were isolatedfrom spontaneously occurring dark sectors of colonies (42). Determina-tions of karyogamy efficiency and nuclear fusion were conducted on bi-lateral matings set up according to the method of Sherwood and Bennett(9). To quantitate nuclear fusion in the zygotes, a small number of matingcells were suspended in phosphate-buffered saline (PBS) following 36 h ofincubation at room temperature. Cells were fixed with paraformaldehydeat a final concentration of 2% for 30 min at room temperature with cul-ture rotation and then washed twice with PBS, and the nuclei were visu-alized with DAPI (4=,6-diamidino-2-phenylindole). To determine themating type loci, individual zygotes were isolated using a micromanipu-lator. After incubation at room temperature for several days, germinated

zygotes were then struck to single colonies on YPD. A single representativecolony from each zygote was analyzed by PCR amplification of the MTLaand MTL� mating type loci, using primer pairs P48/P49 and P50/P51,respectively.

Microscopy. Live-cell microscopy was conducted using a NikonTE200 inverted epifluorescence microscope with a 60 (1.40 numericalaperture [NA]) oil-immersion objective controlled by Metamorph soft-ware. Logarithmically growing cultures were concentrated by a brief cen-trifugation, and cells were pinned in a small volume between a slide andcoverslip. To image nuclei of live cells, 1.0 ml of logarithmically growingculture was concentrated by centrifugation, resuspended to 100 �l, andmixed with Hoechst 33342 (Sigma) to a final concentration of 25 �g ml�1.Cells were incubated with rotation at room temperature for 1 h beforeimaging. Images were false colored in ImageJ (NIH) and contrast adjustedin Photoshop CS5 (Adobe).

Time-lapse imaging of CaTub2-GFP was conducted using a Zeiss AxioObserver.Z1 microscope equipped with a 63 Plan Apo (1.4 NA) lens, a1.6 Optovar lens, a Colibri LED light source, an AxioCam hRM camera,a Pecon environmental chamber, and a model XL S heating unit. Imageswere captured using AxioVision 4.8 software. Cells were immobilizedbetween agarose pads and glass coverslips. Briefly, SDC medium contain-ing 2% agarose was heated to 100°C for 5 min and mixed well. Twenty-fivemicroliters of hot medium-agarose was pipetted onto a microscope slideand quickly flattened to a thin, 1-cm-wide disk by using a second slide.Once the agarose had solidified, the second slide was carefully slid off thepad. One microliter of cell suspension was pipetted on top and coveredwith a 22-mm coverslip. The edges of the coverslip were then sealed usingVALAP (1:1:1 mixture of petrolatum [Vaseline], lanolin, and paraffin).For “short” time lapses, images were captured at 2-min intervals in fivez-slices spaced 0.8 �m apart for a duration of 60 to 90 min. The exposuretime was 500 ms at 25% LED intensity. The temperature was maintainedat 30°C during image acquisition. For “long” time lapses of one or morefull cell cycles, images were captured similarly, but with a 50- to 75-msexposure and a 25% LED intensity for 4 to 6 h. Maximum-intensity pro-jection of z-stacks was conducted using ImageJ.

Anti-tubulin immunofluorescence was conducted as described previ-ously (43). Fixed cells were incubated with a 1:250 dilution of rat anti-�-tubulin antibody (YOL1/34; Santa Cruz) followed by a 1:500 dilution ofAlexa 488-conjugated goat anti-rat secondary antibody (Cell Signaling),both for 1 h at room temperature.

Cloning, protein expression, and protein purification. cDNAs forfull-length CaKAR3 and CaCIK1 were amplified from C. albicans genomicDNA (ATCC 10231D-5), inserted into the TOPO cloning vector (Invit-rogen), and sequenced to verify their identity. For a list of oligonucleo-tides used in this part of the study, please refer to Table S2 in the supple-mental material. Truncated constructs of CaKAR3 and CaCIK1 were thenamplified by PCR and cloned into separate protein expression vectors.CaKAR3 constructs were cloned into pET24d (Novagen) by using NcoIand NotI restriction sites for untagged protein expression. CaCIK1 con-structs were cloned into a modified pET16brTEV (Novagen) vector thatincorporates an N-terminal 10 His tag and a recombinant tobacco etchvirus (rTEV) proteolytic cleavage site by using BamHI and NdeI. All vec-tors were expressed in Escherichia coli BL21-CodonPlus(DE3)-RIL cells(Stratagene) for protein expression in Luria-Bertani (LB) medium sup-plemented with appropriate antibiotics, as previously described (44). Inthe case of the CaKar3/Cik1 heterodimer-forming constructs, both ex-pression vectors were cotransformed into the same expression cell lineand placed under selection with kanamycin, ampicillin, and chloram-phenicol. Cells expressing the recombinant kinesins were harvested bycentrifugation and lysed by flash freezing and sonication, and purificationof all constructs was performed at 4°C.

The CaKar3 motor domain (CaKar3MD) was purified by ion-ex-change chromatography (DEAE SP Sepharose Fast Flow column; GEHealthcare) as described previously (23). The CaCik1 motor homologydomain (CaCik1MHD) was purified by nickel-nitrilotriacetic acid-aga-

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rose (Ni-NTA-agarose) (Qiagen) affinity chromatography as previouslydescribed (20). CaKar3/Cik1 heterodimers were purified using Ni-NTAaffinity chromatography to select for the His-tagged CaCik1 subunit, fol-lowed by size-exclusion chromatography to isolate stable heterodimers,using a Hi-Load 26/60 Superdex 200 prep-grade size-exclusion column(GE Healthcare) in column buffer (20 mM HEPES, pH 7.2, 150 mM NaCl,1 mM MgCl2, 1 mM dithiothreitol [DTT], 0.2 mM ATP, and EDTA-freeprotease inhibitors [Roche]). The stoichiometry of the CaKar3/Cik1 com-plexes was initially estimated by analytical size-exclusion chromatography(Superdex 200 10/300 GL column; GE Healthcare), using a flow rate of 0.5ml min�1 and an injection volume of 1 ml. This column was calibratedwith globular proteins of known molecular sizes (thyroglobulin, 670,000;�-globulin, 158,000; ovalbumin, 44,000; myoglobin, 17,000; and vitaminB12, 1,350) (Bio-Rad). For all protein constructs, final peak fractions fromsize-exclusion chromatography were pooled and concentrated with Ami-con Ultra concentrators (Millipore), and aliquots (30 �l) were flash fro-zen in liquid nitrogen and stored at �80°C.

Sedimentation velocity analytical ultracentrifugation. The stoichi-ometry of the CaCik1L1, CaCik1L2, CaKar3L1, and CaKar3L2 subunitsin the coexpression samples was evaluated by sedimentation velocity anal-ysis, which was performed at 20°C in a Beckman Optima XL-I analyticalultracentrifuge. Samples to be analyzed by analytical ultracentrifugationwere first dialyzed extensively against 20 mM HEPES buffer (pH 7.2)containing 150 mM NaCl, 1 mM MgCl2, 1 mM DTT, 0.1 mM ATP, and0.1 mM EGTA. A 400-�l sample of the CaKar3L1/CaCik1L1 orCaKar3L2/CaCik1L2 complex at 5 �M was loaded into a sample cell con-taining a 2-sector Epon-charcoal centerpiece with a 12-mm optical pathand housed in an An-60 Ti rotor. An aliquot of dialysis buffer was savedfor use in the reference sector of the sample cell. Sedimentation behaviorresulting from a rotor speed of 72,576 g was observed using 400 con-centration gradient scans recorded at 90-s intervals by the XL-I interfer-ence optics. The 400 scans obtained were fitted according to the continu-ous c(S) Lamm equation model in the SEDFIT software package (version9.4) (45) in order to obtain the relative prevalence of each oligomericspecies.

Microtubule-motor equilibrium binding assays. The microtubulebinding affinity of the purified C. albicans motors was determined as de-scribed previously (20), with the following modifications. Reaction mix-tures containing 100 �l microtubules (0 to 6 �M) were incubated with 4�M motor and either 2 mM MgAMPPNP or 2 mM MgADP for 15 min atroom temperature in ATPase buffer (20 mM HEPES, 5 mM magnesiumacetate, 0.1 mM EGTA, 0.1 mM EDTA, 25 mM potassium acetate, 1 mMDTT, 40 �M paclitaxel [Taxol], pH 7.2). For the monomericCaCik1MHD construct, ATPase buffer was supplemented with 150 mMNaCl to minimize any nonspecific protein aggregation that could be mis-interpreted as microtubule-dependent cosedimentation. Reaction mix-tures were sedimented by centrifugation at 312,530 g in a TLA100 rotorfor 15 min at 25°C. Supernatant and pellet fractions were analyzed bySDS-PAGE and visualized with Coomassie brilliant blue R-250. The bandintensities for the pellet fractions were quantified by densitometry, usingImageJ. The amount of kinesin-microtubule complex was plotted as afunction of the microtubule concentration and fit to the following qua-dratic equation in Sigmaplot: (MT · E)/E � 0.5(E0 � Kd,MT � MT0) �[(E0 � Kd,MT � MT0)2 � (4E0MT0)1/2], where MT · E is the amount ofkinesin that sedimented with the microtubule (MT) in the pellet, E0 is thetotal amount of kinesin, Kd,MT is the dissociation constant, and MT0 is theMT concentration.

Motility assays. Motility assays were performed in an acid-washedperfusion chamber by using an oxygen scavenging mix (OSM) (23). Mo-tility assays were performed using CaKar3/Cik1 complexes. Rhodamine-labeled bovine tubulin (X-rhodamine; Cytoskeleton Inc.) was mixed withunlabeled tubulin purified from bovine brain at a molar ratio of 1:4, andthe mixture was polymerized, centrifuged, and resuspended in BRB80 [80mM piperazine-N,N=-bis(2-ethanesulfonic acid) (PIPES), pH 6.8 (KOH),1 mM MgCl2, 1 mM EGTA] and 40 �M paclitaxel. Polarity-marked mi-

crotubules were made by adding green fluorescent tubulin (HiLyte Fluor488; Cytoskeleton Inc.) to microtubule assembly reaction mixtures con-taining red fluorescent microtubule seeds (X-rhodamine; CytoskeletonInc.) according to a previously described protocol (46). Anti-His antibody(final concentration of 1.5 �g ml�1) was applied to the perfusion chamberand incubated for 5 min to allow binding to the glass surface. The chamberwas then washed twice with OSM-0 buffer (1 BRB80, 1.5 mM magne-sium acetate, 5 mg ml�1 casein, 200 �g ml�1 glucose oxidase, 175 �g ml�1

catalase, 25 mM glucose, 2 mM �-mercaptoethanol). The His-taggedCaKar3L1/CaCik1L1 or CaKar3L2/CaCik1L2 kinesin motor was dilutedto 50 nM to 90 nM in OSM-0, added to the chamber, and incubated for 5min to allow binding. Microtubules were then diluted 20-fold in OSM-1(1 OSM-0, 1.5 mM AMPPNP, 40 �M paclitaxel) and incubated for 5min. The perfusion chamber was washed twice with OSM-2 (1 OSM-0,1.5 mM ATP, 40 �M paclitaxel, 0.3 �g �l�1 phosphocreatine kinase, 2mM phosphocreatine) to remove any unattached microtubules. Microtu-bule movement was visualized by taking images every 15 s for a period of10 min, using an Olympus IX-81 inverted microscope with both spin-ning-disk confocal and total internal reflection fluorescence (TIRF) im-aging capabilities (Quorum Technologies Inc.). Quorum WaveFX Meta-morph software (Quorum Technologies Inc.) was used to process theimages and compile them into movies. Microtubule movement wastracked using Image-Pro Plus 6 (Media Cybernetics Inc.).

Steady-state ATPase activity. The microtubule-stimulated steady-state kinetics of CaKar3MD, CaCik1MHD, the CaKar3L1/CaCik1L1complex, and the CaKar3L2/CaCik1L2 complex were determined usingan enzyme-coupled assay as previously described (44, 47). Microtubuleconcentration-dependent reaction mixtures were assembled in A25 buf-fer [25 mM N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), 2 mMmagnesium acetate, 2 mM EGTA, 0.1 mM EDTA, 1 mM �-mercaptoeth-anol, pH 6.9] containing 1 mM Mg-ATP, 2 mM phosphoenolpyruvate,250 �M NADH, 60 �g ml�1 pyruvate kinase, 60 �g ml�1 lactate dehy-drogenase, 0 to 8 �M microtubules, and 200 nM motor in a total volumeof 150 �l. ATP concentration-dependent reaction mixtures were assem-bled in A25 buffer containing 0 to 600 �M Mg-ATP and 1 �M microtu-bules in a total volume of 150 �l. The �A340 s�1 was monitored for eachreaction and converted to the molar concentration of NADH per secondby using a εNADH,340 nm value of 6,220 M�1 cm�1. The ATPase rate permotor head was calculated in units per second by dividing the change inNADH concentration per second by the concentration of the motor. Mi-crotubule and ATP concentrations were plotted against ATPase rates, andKm, K1/2,MT (microtubule concentration required for half-maximalATPase activation), and kcat values were determined by hyperbolic fits ofthe plots of turnover rates against substrate concentrations to the Michae-lis-Menten equation, using SigmaPlot.

Synteny analysis. Genome sequences and annotation for C. albicansSC5314 and S. cerevisiae S288C were retrieved from the Candida GenomeDatabase (http://www.candidagenome.org) and the Saccharomyces Ge-nome Database (http://www.yeastgenome.org). The sequences and loca-tions of CDS features of C. albicans chromosome 3 and S. cerevisiae chro-mosomes XIII and XIV were extracted using Artemis Genome Browser(48). High-scoring sequence pairs were detected for each pair of chromo-somes by using tblastx (NCBI BLAST 0.2.2.29). tblastx was run using thedefault settings, with the exception of a “-db_gencode 12” flag to accountfor the nonstandard genetic code of C. albicans, where required, and the“-outfmt 6” command to generate a tabular output. Chromosome com-parisons were conducted using Artemis Comparison Tool (49).

RESULTSIdentification of a Cik1-related protein in Candida albicans. TheCandida Genome Database (http://www.candidagenome.org/)classifies orf19.306 (NCBI gene ID 3638398) as “uncharacterized”and describes its product as a “putative type II myosin heavychain.” orf19.306 resides between orf19.305 (encoding a protein ofunknown function; upregulated in a cyr1 null mutant) and HFI1



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on chromosome 3 (see Fig. S1A in the supplemental material).The orf19.306 locus shows almost no gene order conservation withS. cerevisiae, with the exception that linkage of C. albicansorf19.306 and HFI1 is conserved for the VIK1 locus on S. cerevisiaechromosome XIV. ScCIK1 does not exhibit this linkage, yet it is aparalog of ScVIK1 and resides in a small cluster of syntenic genesthat resulted from an ancient whole-genome duplication subse-quent to the divergence of C. albicans (50). ClustalW2 sequencealignment of the product of orf19.306 shows a modest 11.6%shared sequence identity with S. cerevisiae Cik1 and a 12.3% iden-tity with S. cerevisiae Vik1 (see Fig. S1B) (51). The highest similar-ity between these three proteins is found at the junction of thehelical “neck” and the C-terminal motor homology domain(MHD). In kinesins, this region is the determinant of motility anddirectionality along microtubules (52). Mutation of this segmentin Candida glabrata Vik1 inhibits the catalytic function of its part-ner Kar3 subunit (53). Similarly to ScCik1, but not ScVik1, theorf19.306 product has a strong nuclear localization signal(NLS), which cNLS Mapper identified between Asp37 and Gly47

(DPLKKRRNSLG) (54). ScCik1 and the product of orf19.306 arealso alike in that they are missing regions of the MHD found inScVik1 that form the microtubule binding cluster (L8a, �5a, L8b,�5b, and L12) and the nucleotide binding pocket (L9) in allknown catalytic kinesins. Like ScCik1 and ScVik1, the orf19.306product also contains a large predicted coiled-coil-forming regionthat may mediate dimerization with the coiled-coil-forming re-gion of CaKar3 (see Fig. S1C). On the basis of these features, wedesignated this protein CaCik1.

CaCik1 and CaKar3 form a heterodimer with minus-end-directed motility. To show that CaCik1 and CaKar3 interact viatheir coiled-coil-forming regions, we cloned and coexpressed thetwo proteins in Escherichia coli and assessed complex formation byaffinity capture chromatography. As with most kinesins, bacterialexpression of full-length CaCik1 and CaKar3 constructs yieldedvery small amounts of protein (data not shown). Therefore, threedifferent pairs of truncated constructs were designed to includecomparably sized sections of the CaCik1 and CaKar3 coiled-coilregions as predicted by COILS analysis (Fig. 1A) (55). By fusing anN-terminal polyhistidine tag to all CaCik1 constructs, we couldperform Ni-NTA affinity purification to easily identify CaCik1-CaKar3 association in cell lysates. SDS-PAGE analysis of the Ni-NTA elution fractions showed that the two longest construct pair-ings, i.e., CaKar3L1 (58.7 kDa) with CaCik1L1 (47.1 kDa) andCaKar3L2 (51.8 kDa) with CaCik1L2 (41 kDa), coeluted, withbands appearing at their calculated molecular masses (Fig. 1B).Conversely, it seems that the CaKar3L3 (44.8 kDa) and CaCik1L3(33.5 kDa) constructs did not contain enough of the coiled-coil-forming sequence to form a stable interaction, because onlyCaCik1L3 was detected in the eluate.

In order to ascertain the stoichiometries of the complexesformed by the two longest CaKar3 and CaCik1 constructs, sedi-mentation velocity analysis was conducted on the Ni-NTA elutionfractions after further purification by analytical size-exclusionchromatography. Figure 1C shows that at a protein concentrationof 5 �M, 81% of the CaKar3L1/CaCik1L1 sample had a sedimen-tation coefficient (S) of 3.81, which corresponds to a molecularmass of 106 kDa. A similar proportion of the CaKar3L2/CaCik1L2sample had a sedimentation coefficient (S) value of 4.05, corre-sponding to a molecular mass of 90 kDa. This indicates that eachcomplex contained one molecule of CaKar3 and one molecule of

CaCik1. Both of these complexes exhibited motility in microtu-bule gliding assays (see Movies S1 and S2 in the supplementalmaterial), with CaKar3L1/CaCik1L1 and CaKar3L2/CaCik1L2complexes displaying mean velocities of 2.01 � 0.03 �m min�1

and 1.34 � 0.03 �m min�1, respectively (Fig. 1D). To determinethe direction bias of this movement, polarity-marked microtu-bules were assembled to differentiate the plus and minus ends andwere then analyzed for microtubule gliding on CaKar3L1/CaCik1L1-coated coverslips (Fig. 1E). For the predominance ofmicrotubules in the field of view that showed a longer green fluo-rescent extension marking the plus end, we observed that theseends were leading. This indicates that CaKar3/Cik1 exhibits mi-nus-end-directed motility.

CaCik1 is required for CaKar3 localization to spindle polebodies. In previous studies, GFP-tagged CaKar3 showed SPB lo-calization (9). To determine whether CaCik1 localizes to the samestructure, a strain expressing CaCik1 with a C-terminal YFP tagwas constructed and expressed with RFP-tagged tubulin (Tub2-RFP). In interphase cells, where short spindles are present and inthe focal plane, one or two CaCik1-YFP foci can be observed (Fig.2A). SPB localization of CaCik1-YFP is also visible on the ends ofanaphase spindles of all lengths, as well as on the SPBs of cells thathave completed anaphase and disassembled their spindles. More-over, a faint but uniform fluorescence signal could sometimes bedetected along the spindles of metaphase and early anaphase cells.As spindle elongation progresses, this spindle-localized popula-tion may become too dilute to be observed above the backgroundautofluorescence. Figure 2B shows that the localization ofCaCik1-YFP is very similar to that of CaKar3-GFP. The occasionalimperfect colocalization of short spindle poles and SPB-localizedCaCik1 or CaKar3 fluorescence in some cells can be attributed tothe dynamic movement of these structures during image acquisi-tion.

We next sought to determine whether CaCik1 and CaKar3localizations during vegetative growth are mutually dependent.Accordingly, a strain expressing CaKar3-GFP was constructed in acik1�/� background, and a strain expressing CaCik1-YFP wasconstructed in a kar3�/� background. Figure 2C shows that theloss of either CaCik1 or CaKar3 led to dissociation of the otherprotein from the spindle poles. Adding a wild-type CaCIK1 alleleback into the Kar3-GFP cik1�/� strain or wild-type CaKAR3 intothe CaCik1-YFP kar3�/� strain restored SPB localization of thefluorescent fusion constructs. In S. cerevisiae, loss of either ScVik1or ScCik1 only partially dissociates ScKar3 from spindle structures(15, 24, 26). The lack of SPB or spindle localization by CaKar3 ina cik1�/� background supports the absence of a cryptic Vik1-likeprotein in C. albicans.

CaCik1 binds microtubules but lacks ATPase activity. Al-though the amino acid sequence of CaCik1 shows major differ-ences in regions that align with the microtubule-binding elementsof kinesin motor domains (i.e., L8a, �5a, L8b, �5b, and L12) (56–58), the dependence of CaKar3 on CaCik1 for spindle localizationcould be explained in part by an involvement of CaCik1 in micro-tubule binding. To investigate this, we generated monomericCaCik1 and CaKar3 constructs consisting of the C-terminal mo-tor (homology) domains (Fig. 3A) and then characterized theirmicrotubule binding affinities in parallel with the dimericCaKar3L2/CaCik1L2 construct, which exhibited higher solubilitythan the longer CaKar3L1/CaCik1L1 construct. Affinity measureswere performed by cosedimentation assays in the presence of ADP

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A B

C

D E

75kD

50kD

37kD

25kD

20kD

CaKar3L1/CaCik1L1

CaKar3L2/CaCik1L2

CaKar3L3/CaCik1L3

1 64 345 687Full Length CaKar3

172 345 687CaKar3L1

CaKar3L2

CaKar3L3229 345 687

291 345 687

CaCik1L1

CaCik1L2

CaCik1L3

Full Length CaCik11 131 619410

242 61941010 x His

295 61941010 x His

356 61941010 x His

0.5

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1.5

2

2.5

3

3.5

4

4.5

5

0 2 4 6 8 10 12 14 16 18 20

10X His Tag Cik1

Kar3

CaKar3L1 = 58.7 kDCaCik1L1 = 47.1 kD Heterodimer (1:1) = 105.8 kD

106kD

212kD

Sedimentation Coefficient [S]

c(S)

60.2kD

336kD

10X His Tag Cik1

Kar3

CaKar3L2 = 51.8 kDCaCik1L2 = 40.9 kDHeterodimer (1:1) = 92.7 kD

90kD

Sedimentation Coefficient [S]

0 2 4 6 8 10 12 14 16 18 0

205kD352kD 585kD

0.2

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Kar3Kar3

Cik1

Cik1Cik1

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35

0.5 1 1.5 2 2.5 3 3.5

CaKar3L1/CaCik1L1v = 2.01 ±0.03 μm/min n = 100

CaKar3L2/CaCik1L2v = 1.34 ±0.03 μm/minn= 84

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Kar3/Cik1

Anti-His Antibody

0 s

225 s

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Velocity (μm/min)

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tail stalkmotor homology

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Sedimentation Coeffff icient [S]

0 2 4 6 8 10 12 14 16 18 20

205kD352kD 585kD

0.2

Kar3/Cik1

Anti-His Antibody

0 s

225 ss

0 s

225 s

---

FIG 1 Structural and functional analyses of CaKar3/Cik1 complexes. (A) Domain architectures and boundaries of the full-length CaKar3 and CaCik1 constructsused for the study. (B) SDS-PAGE analysis of elution fractions following CaKar3/Cik1 Ni-NTA copurification. (C) Sedimentation velocity analysis of CaKar3L1/CaCik1L1 and CaKar3L2/CaCik1L2 complexes. (D) Histograms of the velocity distribution of gliding microtubules in the presence of CaKar3L1/CaCik1L1 orCaKar3L2/CaCik1L2. Time-lapse movies were obtained during 2 to 4 independent experiments (see Movies S1 and S2 in the supplemental material for samplesof these). (E) Illustration depicting polarity-marked microtubules composed of rhodamine-tubulin seeds (red) and Alexa 488-tubulin extensions (green) in aCaKar3/Cik1-driven microtubule gliding assay. Cropped frames showing two of these microtubules (out of a pool of 41 with extended green ends) are shown at0 s and 225 s. The direction of displacement shows the movement of microtubules, with the plus end leading. Corresponding kymographs of the movingmicrotubules are also shown.


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(the weaker binding state for most motile kinesins) or AMPPNP(a nonhydrolyzable ATP analog often used to promote a high-affinity state) (59–61). This analysis showed that CaCik1 binds tomicrotubules independently of CaKar3 (Fig. 3B). Although the

overall proportion of CaCik1 binding was much lower than that ofCaKar3 binding (Fig. 3C), the Kd,MT values for the microtubule-binding population of CaCik1MHD (0.08 �M and 0.16 �M in thepresence of ADP and AMPPNP, respectively) indicate a higher

A Kar3-GFP Tub2-RFPCik1-YFP Tub2-RFPG1/SG2/M Anaphase G1/SG2/M Anaphase

Hoechst

GFP/YFP

RFP

Merged

C Kar3-GFP

CIK1+/+

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cik1Δ/Δ +vector

cik1Δ/Δ +CIK1+

Cik1-YFP

KAR3+/+

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kar3Δ/Δ +KAR3+

B

FIG 2 CaCik1 and CaKar3 localize to SPBs in a mutually dependent manner. CaCik1-YFP (A) and CaKar3-GFP (B) localize to spindle pole bodies in both earlyand late anaphase, as well as in cells that have completed DNA segregation and initiated budding (M/G1). Cells shown are from logarithmically growing culturesof the Kar3-GFP Tub2-RFP (CF100) and Cik1-YFP Tub2-RFP (CF075) strains in SDC medium at 30°C. Tub2-RFP fluorescence and Hoechst staining are shownfor visualization of spindle and nuclear morphologies, respectively. Bars, 5 �m. (C) SPB localizations of CaCik1 and CaKar3 are mutually dependent. Logarith-mically growing cultures of the Cik1-YFP (CF069), Cik1-YFP kar3�/� (CF068), Cik1-YFP kar3�/� � vector (CF150), Cik1-YFP kar3�/� � KAR3� (CF153),Kar3-GFP (CF051), Kar3-GFP cik1�/� (CF056), Kar3-GFP cik1�/� � vector (CF144), and Kar3-GFP cik1�/� � CIK1� (CF147) strains were imaged asdescribed for panel A. The YFP/GFP fluorescence panels (right) are inverted to better show SPB localization, or the lack thereof. Bars, 5 �m.

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affinity than that of CaKar3MD (Kd,MT � 2.03 �M with ADP and1.78 �M with AMPPNP) (Fig. 3D). Moreover, the microtubuleaffinity of the CaKar3L2/CaCik1L2 complex was slightly higher inthe presence of ADP (Kd,MT � 0.82 �M) than in the presence ofAMPPNP (Kd,MT � 1.6 �M). As this affinity was also higher thanthat of the monomeric ADP-bound CaKar3MD construct, it ap-pears that CaCik1 may tether the CaKar3/Cik1 complex to themicrotubules in the ADP-bound state of CaKar3. If the modelsproposed for motility of other Kar3/Cik1 and Kar3/Vik1 motorsare relevant to CaKar3/Cik1, conversion to the ATP-bound statepresumably reverses this interaction toward a CaKar3-bindingmode (20, 62–64).

Two aspects of our data on the steady-state microtubule-stim-ulated ATP turnover kinetics of CaKar3/Cik1 lend support to thishypothesis (see Fig. S2 in the supplemental material). The first isthat CaCik1 lacks ATPase activity, which is also absent in ScCik1,ScVik1, and C. glabrata Vik1 (20, 62–64). The second is thatCaCik1 lowers the concentration of microtubules required to ac-tivate ATP turnover in the CaKar3 subunit. This is evident from acomparison of K1/2,MT values for CaKar3MD and the CaKar3/Cik1 complexes (Table 2), which represent the concentrations atwhich the half-maximal ATPase activity is reached in the absenceand presence of CaCik1. This effect of the Cik1 subunit was alsoobserved for ScKar3/Cik1, ScKar3/Vik1, and the Kar3/Vik1-re-

Tubulin

Tubulin

CaCik1MHD

Pellet

Sup.

CaCik1MHD

TubulinCaKar3MD

TubulinCaKar3MD

TubulinCaKar3L2CaCik1L2

Pellet

Pellet

Sup.

Sup.

MW75kD

37kD

25kD

50kD37kD

50kD

37kD

75kD

50kD

37kD

50kD

37kD

75kD

50kD

37kD

75kD

50kD

25kD

75kD

75kD

Tubulin

MW

MW

μM Tubulin0 0.25 0.5 0.75 2 4 61 1.5

μM Tubulin0 0.25 0.5 0.75 2 4 61 1.5

μM Tubulin0 0.25 0.5 0.75 2 4 61 1.5

CaKar3L2CaCik1L2

Tubulin Concentration (μM)

Frac

tion

of p

r ote

in b

ound

to m

icro

tubu

les

Frac

t ion

of p

rot e

i n b

oun d

t o m

icro

tubu

les

MT Dissociation Constants for Monomeric and Heterodimeric Constructs

Motor Nucleotide Kd,MT [μM]

CaKar3L2/CaCik1L2 ADP 0.82 ± 0.05 CaKar3L2/CaCik1L2 AMPPNP 1.6 ± 0.25 CaKar3MD ADP 2.03 ± 0.36 CaKar3MD AMPPNP 1.78 ± 0.18 CaCik1MHD ADP 0.08 ± 0.02 CaCik1MHD AMPPNP 0.16 ± 0.05

Data reported as mean ± SEM (n=3).

Tubulin Concentration (μM)

C

D

ACaKar3MD

321 345 687

CaCik1MHD399 619410

His

B

N Cmotor domain

motor homology domainN C

0 1 2 3 4 5 60.0

0.2

0.4

0.6

0.8

1.0

CaKar3L2/CaCik1L2 with AMPPNP CaKar3L2/CaCik1L2 with ADP

0 1 2 3 4 5 60.0

0.2

0.4

0.6

0.8

1.0

CaCik1MHD AMPPNP CaCik1MHD ADP

CaKar3MD AMPPNP CaKar3MD ADP

FIG 3 Microtubule binding analysis of CaKar3 and CaCik1. (A) Diagrams of constructs used to give monomeric forms of the CaKar3 motor domain (MD) andthe CaCik1 motor homology domain (MHD). (B) Representative SDS gels of supernatant and pellet fractions recovered from microtubule cosedimentationanalysis with CaCik1MHD and CaKar3MD monomers or the CaKar3L2/CaCik1L2 dimer in the presence of the nonhydrolyzable ATP analog AMPPNP. (C)Microtubule binding curves for CaKar3MD, CaCik1MHD, and the CaKar3L2/CaCik1L2 complex in the presence of 2 mM AMPPNP or ADP. The fraction ofmotor bound to microtubules was plotted against the microtubule concentration and fit to the equation given in Materials and Methods in order to obtain themicrotubule dissociation constants. Error bars show means � standard deviations for three independent experiments. (D) Microtubule (MT) dissociationconstants for monomeric and heterodimeric constructs.



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lated motor from Candida glabrata (20, 64). What still remainsunclear for all forms of Kar3-based motor complexes is how theCik1 or Vik1 subunit binds to microtubules and the precise mech-anism by which the microtubule- and ATP-binding state of theKar3 subunit is communicated to the Cik1/Vik1 subunit to stim-ulate its microtubule release. We and others assume that thecoiled-coil interface is an important component of this commu-nication system (20, 62, 65), which may partly explain the reducedATPase activity and motility rate of the shorter CaKar3L2/CaCik1L2 construct (see Movie S2).

Loss of CaCik1 causes growth and viability defects. Micro-scopic examination of cik1�/� and kar3�/� strains under growthconditions favoring division as blastoconidia showed dramaticchanges in cell morphology (Fig. 4A). Colonies grown on solidmedium showed protruding elongated cells (left panels), and liq-uid cultures of these strains exhibited a mixture of blastoconidiaand cells with long extensions and branching structures resem-bling pseudohyphae (right panels). This type of hyperpolarizedgrowth pattern is characteristic of cell cycle arrest in C. albicans(66). Reintroduction of a wild-type CaCIK1 allele into the cik1�/�strain rescues this growth defect, as does integrating a wild-typeCaKAR3 allele into the RPS1 locus of the kar3�/� strain (data notshown) or at the native locus (9). Quantitation of cell morpholo-gies for each knockout strain showed that approximately 90% ofthe population was in the form of blastoconidia (Fig. 4B), whilemost of the remainder showed a single polarized extension with alength of up to tens of cell diameters. Some of these long polarizedcells had attempted to septate or form another bud (“branched”)either laterally or from the basal cell body. Approximately 1% ofthe total population consisted of cells with multiple branchesand/or constrictions (designated “complex” cells), some of whichhad developed into very large structures.

To assess the generation times of the cik1�/�, kar3�/�, andcombined kar3�/� cik1�/� mutants, logarithmically growingcultures were diluted to 2.5 106 cells per ml in fresh medium,after which their density was measured hourly by using a hemo-cytometer. Figure 4C shows that the cik1�/� and kar3�/� strainsexhibited significantly longer generation times. To further exam-ine the slow-growth phenotype, the colony-forming ability ofeach of the mutants was quantified. This showed that approxi-mately 20% of cells lacking CaCik1 and/or CaKar3 were nonviable(Fig. 4D), indicating that their lower growth rate was not simply aconsequence of delayed progression through the cell cycle. Inter-estingly, only the kar3�/� cells showed a significant growth defectat high temperature (Fig. 4E). This temperature sensitivity wasameliorated by deletion of CaCIK1, indicating that it does notreflect a Cik1-independent Kar3 function. This differs from the

case for S. cerevisiae cik1� and kar3� strains, which are both un-able to grow at 37°C (the edge of this species’ temperature range)(12, 17). However, all C. albicans deletion strains were uniformlysensitive to the ribonucleotide reductase inhibitor hydroxyurea(HU) (Fig. 4F), similar to their S. cerevisiae counterparts.

These findings show that as a result of a partially penetrant cellcycle arrest phenotype in which some cells undergo a switch tohyperpolarized growth, C. albicans cik1�/� and kar3�/� strainsboth have proliferation and viability defects. The observation thatthe phenotype conferred by homozygous deletion of both genessimultaneously is no more severe than that of either single mutantis consistent with these proteins acting in the same complex.

Loss of CaCik1 impairs hyphal growth. Depletion of CaKar3was previously shown to affect invasive filamentous growth bothunder hypha-inducing conditions (Spider medium) and in re-sponse to starvation (extended growth on rich medium) (9). Wefound that cells lacking CaCik1 or both CaCik1 and CaKar3 sharethis phenotype (Fig. 5A). Adding back wild-type copies of CaCIK1or CaKAR3 complemented these knockout phenotypes, althoughthe diameter of the infiltrating hyphal halo was reduced comparedto that of the wild type. This incomplete rescue was presumablydue to integration of the pCIp10-based plasmid at the RPS1 locus,as disruption of one copy of the RPS1 gene is itself known to causemild growth and hypha formation defects (67, 68), and becausewild-type cells with empty vector integration also showed asmaller halo of invasive growth. In the presence of 10% fetal bo-vine serum (FBS), colonies lacking CaKar3 and CaCik1 alsoformed less densely packed crenulations than those of the wildtype, which is another indicator of defective hypha formation (Fig.5A, right panel).

Interestingly, microscopic examination of cells shifted to 37°Cin liquid medium showed that the cik1�/� and kar3�/� mutantswere able to form germ tubes and to undergo a fairly stereotypicalsequence of microtubule-based nuclear movements in the firsthyphal division (Fig. 5B) (69). Both translocation and division ofthe nucleus within the hyphal filament (white arrows) and thereturn of one nucleus to the basal compartment (red arrows) weresimilar to those in wild-type cells. However, when we monitoredhypha formation and septum deposition by calcofluor whitestaining over time, we observed that cells lacking CaCik1 and/orCaKar3 formed hyphal compartments significantly more slowlythan the wild type did (Fig. 5C). Therefore, we propose thatCaCik1 and CaKar3 are essential neither for germ tube formationnor for nuclear division therein. However, in their absence, thedistal hyphal compartment arrests stochastically, terminating ex-tension of the filament. Cells lacking CaCik1 or CaKar3 wouldthus be unable to form complex branching structures or to invadesolid growth media.

CaCik1 is essential for karyogamy. In C. albicans, a stablemating-competent morphological state exists (referred to as the“opaque” state), which is required for cells to undergo matingprojection formation, cell fusion, and karyogamy, i.e., the con-gression and fusion of nuclei from opposite mating types (42).However, instead of undergoing meiosis to produce spores, thetetraploid C. albicans zygote undergoes a series of reductional di-visions whereby chromosomes are lost and the organism returnsto an approximately diploid chromatin content (70, 71). kar3�/�cells exhibit a nuclear congression defect in this specialized matingcycle of C. albicans (11). To determine whether cik1�/� cells arealso unable to undergo nuclear fusion, opaque-phase cells of op-

TABLE 2 Steady-steady ATPase kinetics

Kinesin construct

Value of microtubule-activated ATPase kineticparametera

kcat (s�1) K1/2,MT (�M) Km.ATP (�M)

CaKar3MD 0.46 � 0.03 0.28 � 0.10 43.49 � 6.66CaKar3L1/CaCik1L1 0.46 � 0.01 0.10 � 0.02 46.36 � 7.94CaKar3L2/CaCik1L2 0.27 � 0.01 0.10 � 0.04 69.38 � 9.11a Data are reported as the means � standard errors for the steady-state parametersderived from at least three independent assays. kcat, K1/2,MT, and Km.ATP values werederived from the best fit of the data to the hyperbolae shown in Fig. S2 in thesupplemental material.

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BA

kar3Δ/Δ cik1Δ/Δ 91.1 ± 1 7 ± 0.7 0.7 ± 0.2 0.4 ± 0.2 0.9 ± 0.4

Dividing Arrested Branched Septated Complex

wildtype 99.9 ± 0.1 0.1 ± 0.1

kar3Δ/Δ 87.5 ± 2 9.1 ± 2.2 1.1 ± 0.3 0.9 ± 0.4 1.4 ± 0.7

cik1Δ/Δ 90.7 ± 1.2 6.7 ± 1.3 0.6 ± 0.2 0.9 ± 0.3 1.1 ± 0.6

cik1Δ/Δ + vector 88.8 ± 1.5 9.1 ± 1.7 0.5 ± 0.3 0.2 ± 0.1 1.4 ± 0.4

cik1Δ/Δ + CIK1+ 100

wildtype

kar3Δ/Δ

cik1Δ/Δ

kar3Δ/Δcik1Δ/Δ

cik1Δ/Δ+vector

cik1Δ/Δ+CIK1+

kar3Δ/Δ

cik1Δ/Δ

kar3Δ/Δ cik1Δ/Δ

wildtype

kar3Δ/Δ

cik1Δ/Δ

kar3Δ/Δ cik1Δ/Δ

wildtype

30°C 37°C 42°C 44°C

10 mM HU 15 mM HU 20 mM HU

106 105 104 103

106 105 104 1030%

20%

40%

60%

80%

100%

% V

iabi

lity

kar3Δ/Δcik1Δ/Δ kar3Δ/Δ cik1Δ/Δ

wildtype

** *D

E

F

25C

kar3Δ/Δcik1Δ/Δ kar3Δ/Δ cik1Δ/Δwildtype

0

5

10

15

20

0 1 2 3 4 5 6

Rel

ativ

e C

ells

/ml I

ncre

ase

Time (hours)

wildtype 74.1 ± 1.9Generation time (minutes)

kar3Δ/Δ 111.7 ± 8.1cik1Δ/Δ 98.1 ± 6.0

kar3Δ/Δ cik1Δ/Δ 107.2 ± 8.9

FIG 4 Loss of CaKar3 or CaCik1 affects growth and viability. (A) Left panels show microcolony morphologies of wild-type (CF027), cik1�/� (CF016), cik1�/��vector(CF126), cik1�/� � CIK1� (CF129), kar3�/� (CF024), and kar3�/� cik1�/� (CF019) cells. Cells of the indicated genotype were struck on YPD agar andincubated at room temperature overnight. Bar � 0.1 mm. Logarithmically growing liquid cultures (right panel) show a great degree of morphological hetero-geneity. Bar � 10 �m. (B) Quantification of cell morphology. Dividing, blastoconidia progressing normally through the cell cycle; arrested, cells with a polarextension from the bud, which has not branched or septated; branched, cells which have attempted to produce either a lateral bud from the polar extension ora second bud from the mother cell; septated, cells that have attempted cytokinesis in the polarized extension; complex, cells showing some combination of oneor more constrictions and branches. Data represent the averages for three independent experiments � standard deviations (n 500 cells per strain). (C) KAR3and CIK1 knockouts have a slow-growth phenotype. Logarithmically growing cells in SDC medium were diluted to 2.5 106 cells per ml, incubated at 30°C, andsampled every 60 min. A hemocytometer was used to determine the cell density at each time point. Data points represent the averages for three independentexperiments, with cell density normalized to the starting optical density (OD) for each experiment. Error bars show standard deviations. (Inset) Generation timesof the indicated strains, calculated from the interval of 1 to 6 min. The variations in generation times of cells lacking CaCIK1 and/or CaKAR3 are not statisticallysignificant (Student’s t test; P 0.05) but are different from that of the wild type (P � 0.05). (D) kar3�/�, cik1�/�, and kar3�/� cik1�/� cells exhibit a moderatecell viability defect. Cell densities of late-logarithmic-phase cultures in SDC were determined using a hemocytometer. Cells were serially diluted in sterile PBS,and a known number of CFU were plated in triplicate. Plates were incubated for 2 days at 30°C before colonies were counted. Percent survival was normalizedto that of the wild type, and mean values are presented (n � 6). Error bars represent standard errors of the means (SEM). *, P � 0.01 (Student’s t test). (E) Thekar3�/� strain has a mild, temperature-dependent growth defect that is rescued by the loss of CaCIK1. Logarithmically grown cells were serially diluted to theconcentration indicated, and 5-�l droplets were plated. Plates were incubated for 2 days at the indicated temperature. (F) kar3�/�, cik1�/�, and kar3�/�cik1�/� cells are sensitive to hydroxyurea (HU). Cells were plated as described for panel E and incubated at 30°C for 2 days.



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posing mating types were mixed, deposited on the surface of aSpider medium plate, and allowed to mate and form zygotes for 36hours. A small sample of each mating mixture was then fixed withparaformaldehyde and stained with DAPI to visualize chromatin.Representative zygotes from bilateral wild-type and cik1�/� mat-ings are shown in Fig. S3 in the supplemental material. In wild-type cell mating, 72% of zygotes examined possessed one mass ofchromatin (a single nucleus) (see Fig. S3B). Conversely, only 7%,14%, and 10% of the kar3�/�, cik1�/�, and kar3�/� cik1�/�zygotes, respectively, appeared to possess one chromatin mass.

To determine whether bilateral kar3�/�, cik1�/�, and kar3�/�cik1�/� matings are able to generate tetraploid mating products,individual zygote daughter cells were identified and then micro-

manipulated away from the mating mixture. Subsequently, thesecells were allowed to divide for several days and were thenstreaked to single colonies, one of which was analyzed by PCRfor the presence of MTLa and MTL� mating type loci (9). Themajority (86%) of daughter cells from wild-type MTLa/MTLa MTL�/MTL� matings contained both mating typeloci (see Fig. S3C in the supplemental material), indicating thatkaryogamy occurred to produce these cells. However, no tetra-ploid progeny were identified in cik1�/�, kar3�/�, or kar3�/�cik1�/� matings. Thus, like CaKar3, the CaCik1 protein is es-sential for karyogamy in the C. albicans parasexual cycle.

Disruption of CaCik1 or CaKar3 results in spindle abnor-malities. To investigate the cell cycle arrest phenotype of the

wildtype

wildtype+vector

kar3Δ/Δ

cik1Δ/Δ

kar3Δ/Δcik1Δ/Δ

cik1Δ/Δ+vector

cik1Δ/Δ+CIK1+

kar3Δ/Δ+vector

kar3Δ/Δ+KAR3+

YPD7 Days

Spider5 Daysyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyy yy kar3Δ/Δ cik1Δ/Δ

kar3Δ/Δcik1Δ/Δwildtype

DIC

DAPI

DIC

DAPIO

/N C

ultu

re2

hour

s @

37°

C

YPD+10% FBS3 Days

B

C

0

2

4

6

8

2 4 6 8 10 12

Sep

ta p

er h

ypha

e

Time (hours)kar3Δ/Δ

cik1Δ/Δ kar3Δ/Δ cik1Δ/Δwildtype

A

FIG 5 Loss of CaCik1 disrupts formation of mature hyphae. (A) Cells lacking CaCik1 and/or CaKar3 are unable to form complex hyphal structures on solidmedia. Cells of the indicated genotypes (strains CF027, CF024, CF016, and CF019) were plated onto Spider medium, YPD, or YPD plus 10% FBS and incubatedas indicated before imaging. (B) Cells lacking CaCik1 and/or CaKar3 are able to undergo typical nuclear movement characteristic of the first hyphal division.Stationary-phase cultures were diluted 1:50 into fresh medium and incubated at 37°C to induce hyphae. Following a 2-h incubation, cells were fixed, stained withDAPI, and imaged. White arrows, the first hyphal division occurring in the germ tube; red arrows, postmitotic nuclei that returned to the basal cell body. O/N,overnight; DIC, differential interference contrast. Bar � 10 �m. (C) Growing cik1�/� and kar3�/� hyphae form compartments more slowly than wild-typehyphae. Stationary-phase cells were induced to form hyphae by incubation in 10% FBS at 37°C. Samples were fixed at the indicated times, and cell wall materialwas stained with calcofluor white. The number of septa was counted for at least 30 germ tubes per data point.

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cik1�/�, kar3�/�, and kar3�/� cik1�/� strains, logarithmicallygrowing cultures were fixed and then stained with DAPI to evalu-ate chromatin morphology during budding, mitosis, and division.Blastoconidia were classified as either mononuclear, having chro-matin in the neck, or binucleate and then quantified in terms ofthe proportions of each type in each strain. Polarized cells of thecik1�/� and kar3�/� deletion strains were omitted. Figure 6Ashows that cik1�/� cells, like kar3�/� cells, exhibited a nearly2-fold increase in the proportion of cells with chromatin localiza-tion in the mother-bud neck relative to that for wild-type cells. Anincrease in this population often occurs due to arrest by the spin-

dle assembly checkpoint (SAC) (72). Therefore, we assessed tubu-lin fluorescence patterns in relation to chromatin in logarithmi-cally growing cik1�/�, kar3�/�, and wild-type cells expressing anRFP fusion of �-tubulin (Tub2-RFP). When cells with chromatinspanning the mother-bud neck were classified according to theirspindle characteristics (Fig. 6B), we observed that only 5% ofcik1�/� and kar3�/� cells had elongated (anaphase) spindles(Fig. 6C). For comparison, 40% of wild-type cells contained ananaphase spindle. Strikingly, 50% of the cik1�/� and kar3�/�cells with chromatin spanning the mother-bud neck possessedtwo distinct structures of nuclear Tub2-RFP fluorescence, desig-

wildtype

wildtype + vector

kar3Δ/Δ

kar3Δ/Δ +vector

kar3Δ/Δ +KAR3

kar3Δ/Δ

cik1Δ/Δ

cik1Δ/Δ

cik1Δ/Δ + vector

cik1Δ/Δ +CIK1

0%

20%

40%

60%

80%

100%

Dissociated

Anaphase

Short

AA

DAPI IF: Tubulin Merge

Tub2-RFPcik1Δ/Δ

cik1Δ/Δ

0%

20%

40%

60%

80%

100%

Mononucleate

% o

f Bud

ded

Cel

ls

BinucleateChromatin in Neck

wildtype

Tub2-RFPkar3Δ/Δ

Tub2-RFPcik1Δ/Δ

Tub2-RFP Short

Spindle

Anaphase

Dissociated

Hoechst RFP Merge

% o

f Cel

ls w

ith C

hrom

atin

in N

eck

A B

C

D

Short

Spindle

Anaphase

Dissociated

Short

Spindle

Anaphase

FIG 6 Loss of CaCik1 or CaKar3 causes spindle defects. (A) Blastoconidia of the wild-type (CF027), kar3�/� (CF024), and cik1�/� (CF016) strains were scoredbased on nuclear position. Data represent mean values for three independent replicates of 200 cells for each genotype per replicate. Error bars show SEM. Theproportions of kar3�/� and cik�/� cells with chromatin spanning the neck were significantly different from that of wild-type cells (P � 0.05). (B) Spindlemorphologies of logarithmically growing C. albicans Tub2-RFP (CF057), Tub2-RFP cik1�/� (CF060), and Tub2-RFP kar3�/� (CF058) cells in SDC at 25°C.Bars, 5 �m. (C) Quantitation of nuclear microtubule structures observed in Tub2-RFP � vector (CF132), Tub2-RFP cik1�/� � vector (CF139), Tub2-RFPcik1�/� � CIK1� (CF141), Tub2-RFP kar3�/� � vector (CF135), and Tub2-RFP kar3�/� � KAR3� (CF136) cells in which chromatin extends through themother-bud neck but anaphase has not yet separated the chromatin into two distinct masses. (D) Indirect immunofluorescence (IF) against �-tubulin in cik1�/�(CF016) and Tub2-RFP cik1�/� (CF060) cells shows that dissociated spindle structures are not simply a result of expressing �-tubulin–RFP fusion proteins. Bar,5 �m.



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nated “dissociated” in Fig. 6B and C. This phenotype can be sup-pressed completely by integrating a wild-type allele of CaCik1 intothe cik1�/� background or of CaKar3 into the kar3�/� back-ground, but not by integrating the vector alone (Fig. 6C). More-over, this phenotype is not an artifact of expressing RFP-taggedtubulin, because similar spindle structures were observed incik1�/� cells by using immunofluorescence against �-tubulin(Fig. 6D).

To discern whether these Tub2-RFP structures represent twoshort bipolar spindles in the same nucleus or two monopolar spin-dle halves, we labeled the SPBs in these cells by GFP tagging theprotein encoded by orf19.2600 (NCBI gene ID 3640113), the pu-tative C. albicans homolog of the S. cerevisiae �-tubulin complexmember ScSpc98. As predicted, the product of orf19.2600, namedCaSpc98 herein, localized to spindle poles on both short and elon-gated spindles (Fig. 7A). Examination of cik1�/� cells with ashort, single band of tubulin fluorescence sometimes showed cellswith two visible foci of CaSpc98-GFP, with Tub2-RFP fluores-cence spanning the gap (“bipolar”) (Fig. 7B). However, we alsosaw single or two very closely associated foci of CaSpc98-GFPfluorescence, with nuclear spindle material projecting away in onedirection (“monopolar”). In cells with “dissociated” spindles,each piece possessed a focus of CaSpc98-GFP, indicating thatthese represent dissociated, monopolar half-spindles. In cells thathad switched to hyperpolarized growth (Fig. 7B, bottom panel),these dissociated half-spindles appeared to be drawn apart overtime, resulting in fragmentation of the nucleus (“terminal ar-rest”). These spindle structures are distinct from those observed inS. cerevisiae lacking Cik1 and Kar3, in which spindles are disorga-nized but bipolar (26), indicating a significant functional diver-gence between the kinesin-14 homologs in these two organisms.

cik1�/� spindle defects result from failure to establish spin-dle bipolarity. The monopolar and dissociated spindle structuresobserved by static imaging of cells lacking CaCik1 and CaKar3may have arisen from unstable bipolar spindle structures in meta-phase or anaphase or resulted from an inability to establish thehigh-angle microtubule interactions that precede formation of abipolar spindle. To differentiate between these two possibilitiesand to visualize the formation of the abnormal spindle structures,we utilized time-lapse microscopy to observe cik1�/� cells ex-pressing Tub2-GFP from its native locus. Interestingly, we saw noinstances of anaphase spindles collapsing to monopolar structures(n 50), which argues against a bipolar spindle stability defect inthe cik1�/� mutant. Instead, the spindles of cells lacking CaCik1often appeared to form a tapered structure, which at times wasclearly resolvable as two half-spindles (Fig. 8A). From the patternof microtubule nucleation in images with increased contrast, itappears that both spindle pole bodies were in the “wide” end of thetapered structure (see the images for 8, 10, and 14 min). Over thecourse of the time-lapse experiment, the monopolar spindlesmoved rapidly and erratically around the cytoplasm. Associatedwith this movement was the presence of cytoplasmic microtubulesemanating either parallel to each other, and often toward the bud(20 and 22 min), or in opposite directions (10 and 14 min).

Remarkably, cells with highly mobile monopolar spindles werefrequently able to resolve this structure and to undergo anaphase(Fig. 8B), often after a significant delay. While wild-type cells un-derwent anaphase after an average time of 61.2 � 12.7 min fol-lowing bud formation (n � 18), cik1�/� cells took much longer(mean � 108.8 � 50.2 min; n � 23) and were highly variable

(Fig. 8C). Of the 23 cik1�/� cells tracked during 4- to 6-h time-lapse experiments, 7 showed long, monopolar, highly mobilespindles (green circles) and represented some of the most delayedcells. When cik1�/� cells that we observed budding but not pro-ceeding into anaphase were also considered in our assessment ofbipolar spindle formation, we observed that approximately 64%(21 of 33 cells) formed a spindle with no obvious structural ab-normalities (Fig. 8D; see Movie S3, cell A, in the supplementalmaterial), while 36% (12 of 33 cells) formed a highly mobile mo-nopolar spindle (see Movie S3, cell B). Of the latter cells, just overhalf (7 of 12 cells) were observed to resolve the monopolar spin-dles and to enter anaphase (see Movie S4, cell A). The remainderhad not undergone anaphase by the end of the time-lapse study(see Movie S4, cell B).

Taken together, these findings suggest that bipolar spindle for-mation is compromised in C. albicans cells in which the CaKar3/Cik1 kinesin complex is eliminated and that the monopolar anddissociated half-spindles we observed in cik1�/� and kar3�/�mutants did not result from collapsed anaphase spindles. Based onthe proportion of cells that did not appear to progress to anaphase,we propose that this defect could account for the reduction(20%) (Fig. 4D) in viability of cells lacking this molecularmotor.

DISCUSSION

The suite of microtubule-based motors in hemiascomycete fungicomprises a small complement of kinesins and one dynein (7, 73,74). Among these, the Kar3 kinesin is unique in its partnershipwith a kinesin-like subunit that cannot bind ATP. Like the manyother regulatory mechanisms governing the activity of kinesins,the contributions that these noncatalytic proteins make towardmotor function remain incompletely characterized. Combinedwith the realization that the narrow grouping of eukaryotes har-boring Cik1/Vik1-like proteins includes harmful human patho-gens, such as Candida albicans, this creates a strong rationale foracquiring a deeper understanding of their characteristics andfunctions.

We provide several pieces of evidence showing that orf19.306encodes the interacting partner of C. albicans Kar3 and that thispartner is a homolog of S. cerevisiae Cik1 and Vik1. Although it isnoncatalytic, CaCik1 can bind microtubules and produces a mi-nus-end-directed heterodimeric motor with CaKar3, wherein theCaCik1 subunit has a role in motor localization and catalytic ac-tivity. We also show that some aspects of the phenotype of C.albicans cik1�/� and kar3�/� strains resemble those of S. cerevi-siae knockouts. Intriguingly, though, the dramatic bipolar spindleformation defect we observed in cells lacking CaKar3 or CaCik1has not been reported for S. cerevisiae Kar3, Cik1, or Vik1 mutants(12, 25, 28, 75). Instead, recent work suggests that budding yeaststrains missing ScKar3, ScCik1, or, to a lesser extent, ScVik1 havedifficulty bundling interpolar microtubules (ipMTs) but have in-tact and bipolar spindles (26). Based on these results, the investi-gators of that study proposed a model in which ipMT-boundScKar3/Cik1 engages an ipMT from the other spindle pole with itsmotor heads and then walks toward the minus end to draw the twoipMTs toward an antiparallel arrangement along the spindle axisto form a bipolar spindle. They also proposed that this bundling ofantiparallel microtubules is required for efficient binding of Sc-Cin8 and ScKip1, which work in opposition to ScKar3/Cik1 inproduction of an outward spindle force. Such a mechanism pro-

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Hoechst RFP MergeGFP

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FIG 7 Short spindles and dissociated spindle structures in cells lacking CaCik1 are monopolar. (A) Logarithmically growing C. albicans Spc98-GFP Tub2-RFP(CF160) cells in SDC at 25°C show that the putative Spc98 homolog encoded by orf19.2600 localizes to spindle pole bodies. Bar, 5 �m. (B) cik1�/� Spc98-GFPTub2-RFP (CF163) cells possess many short spindles and separated spindle fragments that are monopolar. Bar, 5 �m.



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vides a plausible explanation for the short spindle length of S.cerevisiae kar3� mutants. Although our imaging of CaSpc98-GFPin C. albicans cells lacking CaCik1 shows SPBs that are often ex-tremely close together, the majority of spindle material is project-ing away from the SPBs rather than bridging the space betweenthem (Fig. 7B, “monopolar” panels). This is inconsistent with thedisorganized yet intact bipolar spindle structures reported byHepperla et al. (26).

From our time-lapse study of the cik1�/� mutants, it appearsthat the bipolar spindle assembly defect manifests from a failure toestablish high-angle interactions between microtubules emanat-ing from newly duplicated spindle poles (76, 77). This would ac-

tivate the spindle assembly checkpoint (SAC), either because sisterkinetochores would not be under bipolar tension or because theywould be attached by microtubules emanating from the samespindle pole (syntelic attachment) (78). The fact that a large pro-portion of cells lacking CaCik1 or CaKar3 are able to overcomethis defect is fascinating and may be mediated by forces exerted onSPBs via cytoplasmic microtubules. These forces may be sufficientto separate the SPBs and reorient the two half-spindles into anantiparallel arrangement, thereby allowing CaKip1 to completebipolar spindle formation and elongation. In instances where abipolar spindle cannot form, separation of the tapered structuresinto two monopolar half-spindles in arrested blastoconidia and

A

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FIG 8 Time-lapse microscopy reveals that CaCik1 is involved in bipolar spindle formation. (A) cik1�/� Tub2-GFP (CF174) cells that failed to establish a bipolarspindle. Each frame is shown at two different contrast levels to help visualize both the spindle structure and the cytoplasmic microtubule position. Monopolarspindle halves remain in a parallel orientation, are moved erratically around the cell, and occasionally nucleate long cytoplasmic microtubules (white arrows).Images are maximum-intensity projections of five 0.8-�m z-slices obtained using a 500-ms exposure time and taken at 2-min intervals. Bars, 5 �m. (B) Parallelmonopolar structures can be resolved to form a bipolar spindle, which undergoes anaphase. Single z-slices from a time-lapse series were captured as describedfor panel A. Bar, 5 �m. (C) Cell cycle progression is generally delayed in cik1�/� Tub2-GFP cells, and anaphase can occur after a prolonged arrest with a parallelmonopolar spindle. Long (4 to 6 h) time-lapse series were captured as described for panel A, but with 50-ms exposures. In cases where both events were observed,the length of time between emergence of the bud and anaphase was measured for Tub2-GFP (CF057) (n � 18) and cik1�/� Tub2-GFP (n � 23) cells. Data forcells that were initially unable to form a bipolar spindle but subsequently underwent anaphase are shown in green. (D) Model of the spindle formation defect ofthe cik1�/� strain. Approximately 64% of observed cell cycles proceeded normally, while 36% went through a period of erratic, monopolar spindle moments. Insome cells, this spindle configuration could be resolved into a bipolar spindle and anaphase could occur.

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the tearing of the nucleus observed in polarized arrested cells(Fig. 7B) could be the results of pulling forces exerted by cytoplas-mic dynein in the absence of a stable antiparallel overlap of ipMTs(79).

The failure of cik1�/� and kar3�/� strains to form extensivehyphal structures is intriguing given that they are able to initiatehyphal filament formation and to complete the stereotypical nu-clear movements of the first hyphal division. Visualization of mi-crotubule structures in hyphal compartments of cik1�/� andkar3�/� cells would help to explain this defect, but this has beenhampered by filamentation growth defects of cells expressingfluorescently labeled tubulin from a native chromosomal locus(80). In addition, attempts to examine microtubules in terminalhyphal compartments by immunofluorescence assay have beenunsuccessful due to a failure of standard methods to permeabilizethe hyphal cell wall and because of the fragility of the fixed fila-ments. Nevertheless, we feel that it is likely that a bipolar spindleformation defect similar to that we described for blastoconidiaexists in cik1�/� and kar3�/� hyphae. Such an event in the ter-minal compartment would halt filament elongation, resulting inthe filamentous growth defect we reported. This is importantfrom a clinical perspective because this morphological transitionis central to the ability of C. albicans to cause tissue destructionand host invasion (81), and it supports the development of Ca-Kar3/Cik1 as a target for drug discovery.

Another point worth considering in this regard is that the lossof CaCik1 results in disruption of karyogamy in C. albicans. In-stead of undergoing meiosis, C. albicans reproduces via a parasex-ual cycle in which two cells of opposite mating types fuse to forma tetraploid zygote, which then returns to an approximately dip-loid state through a loss of chromosomes. Stages of the parasexualcycle are induced by stress, such as interactions with the host im-mune system, and can generate genetic diversity through the cre-ation of aneuploidies, even though mating occurs betweensiblings (82). Although the function of parasexuality in the patho-genesis of C. albicans is still being explored, reducing the avenuesby which this organism can generate diversity in order to evadehost defenses could be a useful side effect of inhibiting the CaKar3/Cik1 complex.

While cells lacking CaCik1 have growth and karyogamy defectssimilar to those seen with the loss of ScCik1, the spindle polelocalization of the CaKar3/Cik1 complex is more like that ofScKar3/Vik1. Genetically, ScKar3/Vik1 appears to function in op-position to ScCin8 and ScKip1 (15), but it is not clear how this isaccomplished mechanistically from the SPB. Unfortunately, addi-tional information about the specific function of SPB-localizedScKar3/Vik1 is limited, so meaningful comparison with the simi-larly localized CaKar3/Cik1 motors is difficult. Other kinesin-14homologs, such as vertebrate XCTK2/HSET and Schizosaccharo-myces pombe Klp2, have been seen to cross-link parallel microtu-bules, which could aid in bipolar spindle formation, particularlyin vertebrates, in which the mitotic spindle is not constrained by aclosed nucleus (83–87). However, these kinesins are prominentlylocalized to the spindle or near kinetochores in vivo rather than tothe spindle poles (83, 88). The Drosophila melanogaster kinesin-14Ncd accumulates at the minus ends between statically cross-linkedparallel microtubules, but it also causes sliding of antiparallel mi-crotubules (89). This indicates an additional function near themidzone. How the loss of microtubule cross-linking activity at thespindle pole of C. albicans cells lacking Kar3 or Cik1 would trans-

late into a defect in plus-end interactions between ipMTs and adefect in bipolar spindle formation is not obvious. Likewise, it isunclear why the minus ends of nuclear microtubules in yeastwould require bundling, as they are nucleated from and tetheredto the SPB by the �-tubulin complex (90). It is possible that theCaKar3/Cik1 complex acts at the SPB to regulate the length andnumber of microtubules in the preanaphase spindle by stimulat-ing microtubule depolymerization. Saunders et al. described thisas a possible function of Kar3 in S. cerevisiae SPBs as a way toestablish normal microtubule arrays, but they suggested that bylate anaphase, ScKar3 is no longer essential in this respect (28).Similarly, we observed that anaphase cells in logarithmicallygrowing cultures lacking CaKar3 and CaCik1 have spindles thatare indistinguishable from those of the wild type in static imagesand that these do not collapse during anaphase. The S. pombekinesin-14 counterpart to ScKar3/Vik1, named Pkl1, also displaysSPB localization (91), and a recent study suggested that S. pombePkl1 and the kinesin-5 homolog Cut7 antagonistically regulatemicrotubule nucleation from the �-TuRC complex (92). This isintriguing because GFP-labeled C. albicans Kip1 (kinesin-5) alsolocalizes prominently to the SPB, with a smaller quantity localiz-ing with the spindle itself (10). Cin8-GFP and Kip1-GFP are alsopredominantly SPB localized in short preanaphase spindles of S.cerevisiae (93). Moreover, S. cerevisiae Vik1 mutants are resistantto the microtubule-depolymerizing drug benomyl (15), suggest-ing that the main function of ScKar3/Vik1 is as a microtubuledepolymerase. If a function of CaKar3/Cik1 in mitosis is to influ-ence minus-end microtubule dynamics, this would require actionfrom the SPB.

The existence of Cik1- and Vik1-like proteins is a curious phe-nomenon. ScCIK1 and ScVIK1 are the result of a whole-genomeduplication (WGD) that occurred approximately 100 to 200 mil-lion years ago (50, 94). The only other kinesin-like proteins tohave persisted as duplicated copies in S. cerevisiae are the kinesin-5motors Kip1 and Cin8, and their functions are intimately tied toboth Kar3 motor complexes (26, 27). Interestingly, in the absenceof all other microtubule-based motor activities (including the ac-tivity of dynein), ScCin8 and ScKar3 alone are able to supportspindle assembly and cell division (75). On the other hand, C.albicans encodes only one kinesin-5 member (CaKip1) and oneCaKar3-based motor, with the sixth member of its kinesin com-plement resembling kinesin-3 (10). In S. cerevisiae, the loss of Kip1and Cin8 simultaneously is lethal, while the loss of Kip1 in C.albicans is not. Therefore, divergence in mitotic kinesin functionmust have occurred since S. cerevisiae and C. albicans divergedhundreds of millions of years ago (95). Interestingly, a BLASTsearch of the genome of Kluyveromyces lactis, a pre-WGD Saccha-romycetes organism, reveals that it possesses the genes for only fivekinesin homologs, only one of which clusters with the S. cerevisiaekinesin-5 proteins in a multiple-sequence alignment. This sug-gests that the ancestral balance of motor activities in the mitoticspindle consisted of a simple motor system involving a single ki-nesin-5 and a single kinesin-14 complex, neither of which is es-sential. This raises questions about the roles of the other motors infungi such as C. albicans and K. lactis.

It is clear that the evaluation of microtubule-based processes inC. albicans as viable drug targets cannot rely solely on the extensiveS. cerevisiae literature. While some roles of the kinesin-14 motorare conserved, there are clear differences in the way that its lossaffects bipolar spindle formation compared to the loss of its S.



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cerevisiae counterparts. Going forward, it will be essential to estab-lish the putative antagonistic actions of other kinesin or dyneinmotors in order to gain a better understanding of the microtubuleinteraction network in C. albicans in general (13, 27, 28). As C.albicans is the pathogen most commonly associated with life-threatening systemic fungal infections, it will also be of great in-terest to learn if the unique kinesin motor complement of C. albi-cans can become a useful target for novel fungicidal drugs.

ACKNOWLEDGMENTS

This work was supported by the National Sciences and Engineering Coun-cil of Canada (grant RGPIN/356025-2013) and the Canadian Institutes ofHealth Research (grant MOP-97832). J.S.A. is a Canada Research Chair(Tier 2) in Structural Biology and an Ontario Early Researcher Awardrecipient.

We thank M. Whiteway for technical assistance with strain construc-tion and S. Zhang, B. Banfield, and P. Young for the use of microscopyfacilities. We also thank K. Munro for assistance with collection and in-terpretation of the analytical ultracentrifugation data for CaKar3/Cik1.

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