Kinesin-related proteins in the mammalian testes: candidate motors for meiosis and morphogenesis.

The kinesin superfamily of molecular motors comprises proteins that participate in a wide variety of motile events within the cell. Members of this family share a highly homologous head domain responsible for force generation attached to a divergent tail domain thought to couple the motor domain to its target cargo. Many kinesin-related proteins (KRPs) participate in spindle morphogenesis and chromosome movement in cell division. Genetic analysis of mitotic KRPs in yeast and Drosophila, as well as biochemical experiments in other species, have suggested models for the function of KRPs in cell division, including both mitosis and meiosis. Although many mitotic KRPs have been identified, the relationship between mitotic motors and meiotic function is not clearly understood. We have used sequence similarity between mitotic KRPs to identify candidates for meiotic and/or mitotic motors in a vertebrate. We have identified a group of kinesin-related proteins from rat testes (termed here testes KRP1 through KRP6) that includes new members of the bimC and KIF2 subfamilies as well as proteins that may define new kinesin subfamilies. Five of the six testes KRPs identified are expressed primarily in testes. Three of these are expressed in a region of the seminiferous epithelia (SE) rich in meiotically active cells. Further characterization of one of these KRPs, KRP2, showed it to be a promising candidate for a motor in meiosis: it is localized to a meiotically active region of the SE and is homologous to motor proteins associated with the mitotic apparatus. Testes-specific genes provide the necessary probes to investigate whether the motor proteins that function in mammalian meiosis overlap with those of mitosis and whether motor proteins exist with functions unique to meiosis. Our search for meiotic motors in a vertebrate testes has successfully identified proteins with properties consistent with those of meiotic motors in addition to uncovering proteins that may function in other unique motile events of the SE.     

Intramanchette transport (IMT): managing the making of the spermatid head,centrosome, and tail

Intramanchette transport (IMT) and intraflagellar transport (IFT) share similar molecular components: a raft protein complex transporting cargo proteins mobilized along microtubules by molecular motors. IFT, initially discovered in flagella of Chlamydomonas, has been also observed in cilia of the worm Caenorhabditis elegans and in mouse ciliated and flagellated cells. IFT has been defined as the mechanism by which protein raft components (also called IFT particles) are displaced between the flagellum and the plasma membrane in the anterograde direction by kinesin-II and in the retrograde direction by cytoplasmic dynein 1b. Mutation of the gene Tg737, encoding one of the components of the raft protein complex, designated Polaris in the mouse and IFT88 in both Chlamydomonas and mouse, results in defective ciliogenesis and flagellar development as well as asymmetry in left-right axis determination. Polaris/IFT88 is detected in the manchette of mouse and rat spermatids. Indications of an IMT mechanism originated from the finding that two proteins associated with the manchette (Sak57/K5 and TBP-1, the latter a component of the 26S proteasome) repositioned to the centrosome and sperm tail once the manchette disassembled. IMT has the features of the IFT machinery but, in addition, facilitates nucleocytoplasmic exchange activities during spermiogenesis. An example is Ran, a small GTPase present in the nucleus and cytoplasm of round spermatids and in the manchette of elongating spermatids. Upon disassembly of the manchette, Ran GTPase is found in the centrosome region of elongating spermatids. Because defective molecular motors and raft proteins result in defective flagella, cilia, and cilia-containing photoreceptor cells in the retina, IMT and IFT are emerging as essential mechanisms for managing critical aspects of sperm development. Details of specific role of Ran GTPase in nucleocytoplasmic transport and its relocation from the manchette to the centrosome to the sperm tail await elucidation.     

KIF1Bbeta2, capable of interacting with CHP,is localized to synaptic vesicles

Kinesin family proteins are microtubule-dependent molecular motors involved in the intracellular motile process. Using a Ca2+ -binding protein, CHP (calcineurin B homologous protein), as a bait for yeast two hybrid screening, we identified a novel kinesin-related protein, KIF1Bbeta2. KIF1Bbeta2 is a member of the KIF1 subfamily of kinesin-related proteins, and consists of an amino terminal KIF1B-type motor domain followed by a tail region highly similar to that of KIF1A. CHP binds to regions adjacent to the motor domains of KIF1Bbeta2 and KIF1B, but not to those of the other KIF1 family members, KIF1A and KIF1C. Immunostaining of neuronal cells showed that a significant portion of KIF1Bbeta2 is co-localized with synaptophysin, a marker protein for synaptic vesicles, but not with a mitochondria-staining dye. Subcellular fractionation analysis indicated the co-localization of KIF1Bbeta2 with synaptophysin. These results suggest that KIF1Bbeta2, a novel CHP-interacting molecular motor, mediates the transport of synaptic vesicles in neuronal cells.     

Myo3A,one of two class III myosin genes expressed in vertebrate retina,is localized to the calycal processes of rod and cone photoreceptors and is expressed in the sacculus

The striped bass has two retina-expressed class III myosin genes, each composed of a kinase, motor, and tail domain. We report the cloning, sequence analysis, and expression patterns of the long (Myo3A) and short (Myo3B) class III myosins, as well as cellular localization and biochemical characterization of the long isoform, Myo3A. Myo3A (209 kDa) is expressed in the retina, brain, testis, and sacculus, and Myo3B (155 kDa) is expressed in the retina, intestine, and testis. The tails of these two isoforms contain two highly conserved domains, 3THDI and 3THDII. Whereas Myo3B has three IQ motifs, Myo3A has nine IQ motifs, four in its neck and five in its tail domain. Myo3A localizes to actin filament bundles of photoreceptors and is concentrated in the calycal processes. An anti-Myo3A antibody decorates the actin cytoskeleton of rod inner/outer segments, and this labeling is reduced by the presence of ATP. The ATP-sensitive actin association is a feature characteristic of myosin motors. The numerous IQ motifs may play a structural or signaling role in the Myo3A, and its localization to calycal processes indicates that this myosin mediates a local function at this site in vertebrate photoreceptors.     

Identification and partial characterization of mitotic centromere- associated kinesin, a kinesin-related protein that associates with centromeres during mitosis

Using antipeptide antibodies to conserved regions of the kinesin motor domain, we cloned a kinesin-related protein that associates with the centromere region of mitotic chromosomes. We call the protein MCAK, for mitotic centromere-associated kinesin. MCAK appears concentrated on centromeres at prophase and persists until telophase, after which time the localization disperses. It is found throughout the centromere region and between the kinetochore plates of isolated mitotic CHO chromosomes, in contrast to two other kinetochore-associated microtubule motors: cytoplasmic dynein and CENP-E (Yen et al., 1992), which are closer to the outer surface of the kinetochore plates. Sequence analysis shows MCAK to be a kinesin-related protein with the motor domain located in the center of the protein. It is 60-70% similar to kif2, a kinesin-related protein originally cloned from mouse brain with a centrally located motor domain (Aizawa et al., 1992). MCAK protein is present in interphase and mitotic CHO cells and is transcribed as a single 3.4-kb message.     

Ran and Calcineurin Can Participate Collaboratively in the Regulation of Spermatogenesis in Scallop

Calcineurin is a calcium/calmodulin-dependent protein phosphatase that plays important roles in the transduction of calcium signals in a variety of tissues. In addition, calcineurin has been implicated in the process of spermatogenesis. A novel calcineurin-binding protein, CaNBP75, has been identified in scallop testis. The C-terminal region of CaNBP75 is homologous to the C-terminal region of RanBP3, a Ran-binding domain-containing protein. A small G protein Ran has been involved in spermiogenesis by virtue of the fact that its localization in spermatids changes during spermiogenesis. The current study was performed to investigate the functions of Ran and CaNBP75 in the regulation of calcineurin in testis to further understand the basic functions of calcineurin during spermatogenesis. First, cloning and sequencing of a scallop Ran cDNA isolated from testis revealed that scallop Ran is well-conserved at the amino acid level. Secondly, direct binding of Ran to CaNBP75 was demonstrated in an in vitro pull-down assay. Thirdly, analysis of the tissue distribution of Ran, CaNBP75, and calcineurin showed that these proteins are abundantly expressed in testis. Fourthly, comparison of the expression profiles of Ran and CaNBP75 with that of calcineurin in scallop testis during the maturation cycle revealed that Ran and CaNBP75 mRNA levels increase during meiosis and spermiogenesis, similar to calcineurin. Finally, co-immunoprecipitation analysis suggests that Ran, CaNBP75, and calcineurin interact in scallop testis during maturation. These results suggest that Ran, CaNBP75, and calcineurin may act in a coordinated manner to regulate spermatogenesis.     

Characterization of the KLP68D kinesin-like protein in Drosophila: possible roles in axonal transport

This paper describes the molecular and biochemical properties of KLP68D, a new kinesin-like motor protein in Drosophila melanogaster. Sequence analysis of a full-length cDNA encoding KLP68D demonstrates that this protein has a domain that shares significant sequence identity with the entire 340-amin acid kinesin heavy chain motor domain. Sequences extending beyond the motor domain predict a region of alpha-helical coiled-coil followed by a globular "tail" region; there is significant sequence similarity between the alpha-helical coiled- coil region of the KLP68D protein and similar regions of the KIF3 protein of mouse and the KRP85 protein of sea urchin. This finding suggests that all three proteins may be members of the same family, and that they all perform related functions. KLP68D protein produced in Escherichia coli is, like kinesin itself, a plus-end directed microtubule motor. In situ hybridization analysis of KLP68D RNA in Drosophila embryos indicates that the KLP68D gene is expressed primarily in the central nervous system and in a subset of the peripheral nervous system during embryogenesis. Thus, KLP68D may be used for anterograde axonal transport and could conceivably move cargoes in fly neurons different than those moved by kinesin heavy chain or other plus-end directed motors.     

Mitotic motors in Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae provides a unique opportunity for study of the microtubule-based motor proteins that participate in mitotic spindle function. The genome of Saccharomyces encodes a relatively small and genetically tractable set of microtubule-based motor proteins. The single cytoplasmic dynein and five of the six kinesin-related proteins encoded have been implicated in mitotic spindle function. Each motor protein is unique in amino acid sequence. On account of functional overlap, no single motor is uniquely required for cell viability, however. The ability to create and analyze multiple mutants has allowed experimental dissection of the roles performed by each mitotic motor. Some of the motors operate within the nucleus to assemble and elongate the bipolar spindle (kinesin-related Cin8p, Kip1p, Kip3p and Kar3p). Others operate on the cytoplasmic microtubules to effect spindle and nuclear positioning within the cell (dynein and kinesin-related Kip2p, Kip3p and Kar3p). The six motors apparently contribute three fundamental activities to spindle function: motility, microtubule cross-linking and regulation of microtubule dynamics.     

Association of the testis-specific TRIM/RBCC protein RNF33/TRIM60 with the cytoplasmic motor proteins KIF3A and KIF3B

The Rnf33/Trim60 gene is temporally transcribed in the preimplantation embryo before being silenced at the blastocyst stage but Rnf33 expression is detected in adult testis of the mouse. The putative RNF33 protein is a tripartite motif (TRIM)/RBCC protein composed of a typical RING zinc finger, a B-box 2, two α-helical coiled-coil segments, and a B30.2 domain. As a first step towards the elucidation of the biologic function of RNF33, we aimed in this study to elucidate proteins that associate with RNF33. RNF33-interacting proteins were first derived by the yeast two-hybrid system followed by co-immunoprecipitation assays. Interacting domains were determined by deletion mapping in genetic and biochemical analyzes. RNF33 was shown to interact with the kinesin-2 family members 3A (KIF3A) and 3B (KIF3B) motor proteins in the heterodimeric form known to transport cargos along the microtubule. Domain mapping showed that the RB and B30.2 domains of RNF33 interacted with the respective carboxyl non-motor domains of KIF3A and KIF3B. Since RNF33 interacted with the carboxyl-terminal tail of the KIF3A-KIF3B heterodimer, the motor head section of KIF3A-KIF3B was free and available for association with designated cargo(s) and movement along the microtubule. Data also suggest that RNF33 most likely interacted with KIF3A-KIF3B independent of the adaptor kinesin-associated protein KAP3. This study is a first demonstration of a TRIM protein, namely RNF33, that interacts with the kinesin molecular motors possibly contributing to kinesin-dependent mobilization of specific cargo(s) along the microtubule in the testis of the mouse.     

A novel actin-bundling kinesin-related protein from Dictyostelium discoideum

Actin filaments and microtubules are two major cytoskeletal systems involved in wide cellular processes, and the organizations of their filamentous networks are regulated by a large number of associated proteins. Recently, evidence has accumulated for the functional cooperation between the two filament systems via associated proteins. However, little is known about the interactions of the kinesin superfamily proteins, a class of microtubule-based motor proteins, with actin filaments. Here, we describe the identification and characterization of a novel kinesin-related protein named DdKin5 from Dictyostelium. DdKin5 consists of an N-terminal conserved motor domain, a central stalk region, and a C-terminal tail domain. The motor domain showed binding to microtubules in an ATP-dependent manner that is characteristic of kinesin-related proteins. We found that the C-terminal tail domain directly interacts with actin filaments and bundles them in vitro. Immunofluorescence studies showed that DdKin5 is specifically enriched at the actin-rich surface protrusions in cells. Overexpression of the DdKin5 protein affected the organization of actin filaments in cells. We propose that a kinesin-related protein, DdKin5, is a novel actin-bundling protein and a potential cross-linker of actin filaments and microtubules associated with specific actin-based structures in Dictyostelium.     

Separation of meiotic and mitotic effects of claret non-disjunctional on chromosome segregation in Drosophila.

The claret (ca) locus in Drosophila encodes a kinesin-related motor molecule that is required for proper distribution of chromosomes in meiosis in females and in the early mitotic divisions of the embryo. Here we demonstrate that a mutant allele of claret non-disjunctional (ca(nd)), non-claret disjunctional Dominant (ncdD), causes abnormalities in meiotic chromosome segregation, but is near wild-type with respect to early mitotic chromosome segregation. DNA sequence analysis of this mutant allele reveals two missense mutations compared with the predicted wild-type protein. One mutation lies in a proposed microtubule binding region of the motor domain and affects an amino acid residue that is conserved in all kinesin-related proteins reported to date. This region of the motor domain can be used to distinguish meiotic and mitotic motor function, defining an amino acid sequence criterion for classifying motors according to function. ncdD's mutant meiotic effect, but near wild-type mitotic effect, suggests that interactions of the ca motor protein with spindle microtubules differ in meiosis and mitosis.     

Fluorescence resonance energy transfer biosensors that detect Ran conformational changes and a Ran x GDP-importin-beta -RanBP1 complex in vitro and in intact cells

The Ran GTPase plays a central role in nucleocytoplasmic transport. Association of Ran x GTP with transport carriers (karyopherins) triggers the loading/unloading of export or import cargo, respectively. The C-terminal tail of Ran x GTP is deployed in an extended conformation when associated with a Ran binding domain or importins. To monitor tail orientation, a Ran-GFP fusion was labeled with the fluorophore Alexa546. Fluorescence resonance energy transfer (FRET) occurs efficiently between the green fluorescent protein (GFP) and Alexa546 for Ran x GDP and Ran x GTP, suggesting that the tail is tethered in both states. However, Ran x GTP complexes with importin-beta, RanBP1, and Crm1 all show reduced FRET consistent with tail extension. Displacement of the C-terminal tail of Ran by karyopherins may be a general mechanism to facilitate RanBP1 binding. A Ran x GDP-RanBP1-importin-beta complex also displayed a low FRET signal. To detect this complex in vivo, a bipartite biosensor consisting of Ran-Alexa546 plus GST-GFP-RanBP1, was co-injected into the cytoplasm of cells. The Ran redistributed predominantly to the nucleus, and RanBP1 remained cytoplasmic. Nonetheless, a robust cytoplasmic FRET signal was detectable, which suggests that a significant fraction of cytoplasmic Ran.GDP may exist in a ternary complex with RanBP1 and importins.     

Single-molecule behavior of monomeric and heteromeric kinesins

Conventional kinesin is capable of long-range, processive movement along microtubules, a property that has been assumed to be important for its role in membrane transport. Here we have investigated whether the Caenorhabditis elegans monomeric kinesin unc104 and the sea urchin heteromeric kinesin KRP85/95, two other members of the kinesin superfamily that function in membrane transport, are also processive. Both motors were fused to green fluorescent protein, and the fusion proteins were tested for processive ability using a single-molecule fluorescence imaging microscope. Neither unc104-GFP nor KRP85/95-GFP exhibited processive movement (detection limit approximately 40 nm), although both motors were functional in multiple motor microtubule gliding assays (v = 1760 +/- 540 and 202 +/- 37 nm/s, respectively). Moreover, the ATP turnover rates (5.5 and 3.1 ATPs per motor domain per second, respectively) are too low to give rise to the observed microtubule gliding velocities, if only a single motor were driving transport with an 8 nm step per ATPase cycle. Instead, the results suggest that these motors have low duty cycles and that high processivity may not be required for efficient vesicle transport. Conventional kinesin's unusual processivity may be required for efficient transport of protein complexes that cannot carry multiple motors.     

KRP/telokin differentially regulates filament assembly and phosphorylation of light chains of non-muscle myosin II in fibroblasts

Transgenic 3T3 fibroblasts have been generated that express either the wild-type KRP or its truncated mutant lacking the C-terminal domain, which primarily contributes to myosin binding of KRP. It was found that KRP-expressing cells display a significantly increased content of myosin filaments and a reduced level of rMLC phosphorylation, whereas the mock transfected cells or cells expressing the C-terminally truncated KRP do not. Our results suggest that (1) KRP promotes the polymerization of myosin II and reduces the rMLC phosphorylation level in cells, (2) KRP acts through direct binding to myosin II, and (3) transgenic 3T3 fibroblasts stably expressing KRP represent a useful and versatile model to study the role of myosin II filament dynamics in cell motility.     

Identification and characterization of a synaptojanin 2 splice isoform predominantly expressed in nerve terminals

We have previously identified synaptojanin 1, a phosphoinositide phosphatase predominantly expressed in the nervous system, and synaptojanin 2, a broadly expressed isoform. Synaptojanin 1 is concentrated in nerve terminals, where it has been implicated in synaptic vesicle recycling and actin function. Synaptojanin 2A is targeted to mitochondria via a PDZ domain-mediated interaction. We have now characterized an alternatively spliced form of synaptojanin 2 that shares several properties with synaptojanin 1. This isoform, synaptojanin 2B, undergoes further alternative splicing to generate synaptojanin 2B1 and 2B2. Both amphiphysin and endophilin, two partners synaptojanin 1, bind synaptojanin 2B2, whereas only amphiphysin binds synaptojanin 2B1. Sequence similar to the endophilin-binding site in synaptojanin 1 is present only in synaptojanin 2B2, and this sequence was capable of affinity purifying endophilin from rat brain. The Sac1 domain of synaptojanin 2 exhibited phosphoinositide phosphatase activity very similar to that of the Sac1 domain of synaptojanin 1. Site-directed mutagenesis further illustrated its functional similarity to the catalytic domain of Sac1 proteins. Antibodies raised against the synaptojanin 2B-specific carboxyl-terminal region identified a 160-kDa protein in brain and testis. Immunofluorescence showed that synaptojanin 2B is localized at nerve terminals in brain and at the spermatid manchette in testis. Active Rac1 GTPase affects the intracellular localization of synaptojanin 2, but not of synaptojanin 1. These results suggest that synaptojanin 2B has a partially overlapping function with synaptojanin 1 in nerve terminals, with additional roles in neurons and other cells including spermatids.     

Telokin/KRP differentially modulates myosin II filament assembly and regulatory light chain phosphorylation in fibroblasts

Transgenic 3T3 fibroblasts were made to express either wild-type telokin (KRP) or its truncated version lacking the C-terminal domain essential for binding to myosin. The content of myosin II filaments was markedly increased while regulatory light chain phosphorylation was decreased in the cells expressing KRP but not the C-truncated version. It could be concluded that (i) KRP promotes polymerization of nonmuscle myosin but reduces its RLC phosphorylation, (ii) these effects involve direct KRP binding to myosin, and (iii) KRP-expressing fibroblasts are a convenient model for assessing the role of myosin structural dynamics in cell motility.     

A novel rat homolog of the Saccharomyces cerevisiae ubiquitin-conjugating enzymes UBC4 and UBC5 with distinct biochemical features is induced during spermatogenesis.

The Saccharomyces cerevisiae ubiquitin-conjugating enzymes (E2s) UBC4 and UBC5 are essential for degradation of short-lived and abnormal proteins. We previously identified rat cDNAs encoding two E2s with strong sequence similarity to UBC4 and UBC5. These E2 isoforms are widely expressed in rat tissues, consistent with a fundamental cellular function for these E2s. We now report a new isoform, 8A, which despite having >91% amino acid identity with the other isoforms, shows several novel features. Expression of the 8A isoform appears restricted to the testis, is absent in early life, but is induced during puberty. Hypophysectomy reduced expression of the 8A isoform. In situ hybridization studies indicated that 8A mRNA is expressed mainly in round spermatids. Immunoblot analyses showed that 8A protein is found not only in subfractions of germ cells enriched in round spermatids but also in subfractions containing residual bodies extruded from more mature elongated spermatids, indicating that the protein possesses a longer half-life than the mRNA. Unlike all previously identified mammalian and plant homologs of S. cerevisiae UBC4, which possess a basic pI, the 8A isoform is unique in possessing an acidic pI. The small differences in sequence between the 8A isoform and other rat isoforms conferred differences in biochemical function. The 8A isoform was less effective than an isoform with a basic pI or ineffective in conjugating ubiquitin to certain fractions of testis proteins. Thus, although multiple isoforms of a specific E2 may exist to ensure performance of a critical cellular function, our data demonstrate, for the first time, that multiple genes also permit highly specialized regulation of expression of specific isoforms and that subtle differences in E2 primary structure can dictate conjugation of ubiquitin to different subsets of cellular proteins.