The kinesin-like ncd protein of Drosophila is a minus end-directed microtubule motor

The Drosophila ncd gene is required for chromosome segregation during female meiosis. Previous analyses suggested that the ncd gene encoded a protein with sequence similarity to the kinesin motor domain, which suggested that, like kinesin, the ncd protein might be a plus end-directed microtubule motor. Here we describe the expression of ncd protein in E. coli and the initial characterization of the ncd protein's motor properties. The ncd protein is indeed a microtubule motor, but the polarity of movement is minus end directed. The ncd protein also has microtubule bundling activity. These findings limit possible models for the in vivo functions of the ncd protein and suggest that motor proteins with similar sequence can generate movement in opposite directions along a microtubule.     

Mitotic HeLa cells contain a CENP-E-associated minus end-directed microtubule motor.

A minus end-directed microtubule motor activity from extracts of HeLa cells blocked at prometaphase/metaphase of mitosis with vinblastine has been partially purified and characterized. The motor activity was eliminated by immunodepletion of Centromere binding protein E (CENP-E). The CENP-E-associated motor activity, which was not detectable in interphase cells, moved microtubules at mean rates of 0.46 micron/s at 37 degrees C and 0.24 micron/s at 25 degrees C. The motor activity co-purified with CENP-E through several purification procedures. Motor activity was clearly not due to dynein or to kinesin. The microtubule gliding rates of the CENP-E-associated motor were different from those of dynein and kinesin. In addition, the pattern of nucleotide substrate utilization by the CENP-E-associated motor and the sensitivity to inhibitors were different from those of dynein and kinesin. The CENP-E-associated motor had an apparent native molecular weight of 874,000 Da and estimated dimensions of 2 nm x 80 nm. This is the first demonstration of motor activity associated with CENP-E, strongly supporting the hypothesis that CENP-E may act as a minus end-directed microtubule motor during mitosis.     

Mechanisms for focusing mitotic spindle poles by minus end-directed motor proteins

During the formation of the metaphase spindle in animal somatic cells, kinetochore microtubule bundles (K fibers) are often disconnected from centrosomes, because they are released from centrosomes or directly generated from chromosomes. To create the tightly focused, diamond-shaped appearance of the bipolar spindle, K fibers need to be interconnected with centrosomal microtubules (C-MTs) by minus end-directed motor proteins. Here, we have characterized the roles of two minus end-directed motors, dynein and Ncd, in such processes in Drosophila S2 cells using RNA interference and high resolution microscopy. Even though these two motors have overlapping functions, we show that Ncd is primarily responsible for focusing K fibers, whereas dynein has a dominant function in transporting K fibers to the centrosomes. We also report a novel localization of Ncd to the growing tips of C-MTs, which we show is mediated by the plus end-tracking protein, EB1. Computer modeling of the K fiber focusing process suggests that the plus end localization of Ncd could facilitate the capture and transport of K fibers along C-MTs. From these results and simulations, we propose a model on how two minus end-directed motors cooperate to ensure spindle pole coalescence during mitosis.     

Identification of a minus end-specific microtubule-associated protein located at the mitotic poles in cultured mammalian cells.

A mitosis-specific centrosomal component was studied with a human autoantibody, SP-H, which immunostained mitotic poles and interphase nuclei, and a single polypeptide with an apparent molecular mass of 200 to 230 kDa in various lines of cultured cells. Early mitotic PtK1 cells treated with 10 micrograms/ml taxol contained short bundles of parallel microtubules around the nuclei and cell periphery. At the time of nuclear envelope breakdown, the nuclear staining by SP-H disappeared, and the antigen relocated at one end of the parallel microtubules. Determination of the microtubule polarity demonstrated that the peripheral bundles of microtubules were arranged with their minus ends directed to the cell periphery, and the SP-H antigen was specifically localized at this end. Parallel microtubules were further rearranged first into a fan-like shape, and then into completely radial structures as observed by De Brabander et al. (Int. Rev. Cytol. 101, 215-274 (1986)). The SP-H antigen was always detected at the minus end domain of such microtubule-containing structures during the transformation process. When microtubules were depolymerized by nocodazole treatment, the SP-H antigen appeared as discrete cytoplasmic foci, suggesting that the antigen may self-associate, forming multimeric structures. The antigen in mitotic HeLa cell extracts co-sedimented in vitro with exogenous brain microtubules. The microtubule-associated SP-H antigen was insensitive to ATP extraction, but was removed from microtubules by treatment with 0.5 M NaCl. Thus the 200 to 230 kDa centrosomal component could be a novel microtubule-associated protein with affinity for the minus end of microtubules, and it might play an essential role in the organization of spindle poles during mitosis.     

KIFC3, a microtubule minus end-directed motor for the apical transport of annexin XIIIb-associated Triton-insoluble membranes

We have identified and characterized a COOH-terminal motor domain-type kinesin superfamily protein (KIFC), KIFC3, in the kidney. KIFC3 is a minus end-directed microtubule motor protein, therefore it accumulates in regions where minus ends of microtubules assemble. In polarized epithelial cells, KIFC3 is localized on membrane organelles immediately beneath the apical plasma membrane of renal tubular epithelial cells in vivo and polarized MDCK II cells in vitro. Flotation assay, coupled with detergent extraction, demonstrated that KIFC3 is associated with Triton X-100-insoluble membrane organelles, and that it overlaps with apically transported TGN-derived vesicles. This was confirmed by immunoprecipitation and by GST pulldown experiments showing the specific colocalization of KIFC3 and annexin XIIIb, a previously characterized membrane protein for apically transported vesicles (Lafont, F., S. Lecat, P. Verkade, and K. Simons. 1998. J. Cell Biol. 142:1413-1427). Furthermore, we proved that the apical transport of both influenza hemagglutinin and annexin XIIIb was partially inhibited or accelerated by overexpression of motor-domainless (dominant negative) or full-length KIFC3, respectively. Absence of cytoplasmic dynein on these annexin XIIIb-associated vesicles and distinct distribution of the two motors on the EM level verified the existence of KIFC3-driven transport in epithelial cells.     

Identification of a kinesin-like microtubule-based motor protein in Dictyostelium discoideum.

Dictyostelium discoideum, a unicellular eukaryote amenable to both biochemical and genetic dissection, provides an attractive system for studying microtubule-based transport. In this work, we have identified microtubule-based motor activities in Dictyostelium cell extracts and have partially purified a protein that induces microtubule translocation along glass surfaces. This protein, which sediments at approximately 9S in sucrose density gradients and is composed of a 105 kd polypeptide, generates anterograde movement along microtubules that is insensitive to 5 mM NEM (N-ethyl-maleimide) but sensitive to 200 microM vanadate, and has similar nucleotide-dependent microtubule binding properties to those of kinesins purified from mammals, sea urchin and Drosophila. This kinesin-like molecule from Dictyostelium, however, is immunologically distinct from bovine and squid neuronal kinesins and supports microtubule movement on glass at four-fold greater velocities (2.0 versus 0.5 microns/sec). Furthermore, AMP-PNP (adenylyl imidodiphosphate), which promotes attachment of previously characterized kinesins to microtubules, decreases the affinity of the Dictyostelium kinesin homolog for microtubules. Thus, an AMP-PNP-induced rigor binding may not be a characteristic of kinesins from lower eukaryotes.     

Localization of a kinesin-like protein in generative cells of tobacco

Summary We have obtained immunofluorescence and immunoblot evidence for the presence of kinesin-like protein (KLP) in pollen tubes of tobacco using an antibody generated against peptides encoded by theKATA gene ofArabidopsis. This antibody recognizes an M_r 140,000 polypeptide inArabidopsis seedlings, and stains the mitotic apparatus in this species as well as in tobacco suspension cells. In tobacco pollen tubes prepared for dual immunofluorescence localizations of KLP and -tubulin, the antibody binds transiently to microtubule (Mt) bundles and the nucleus in premitotic generative cells; it then stains the developing mitotic apparatus as the nuclear envelope breaks down. By metaphase, fluorescence is located over kinetochore fibers and associated Mts. Localization of KLP is concentrated in the midzone during anaphase, and by early cytokinesis, it closely brackets the cell plate. Phragmoplast fluorescence then spreads along the phragmoplast distal to the cell plate. Punctate staining is also detected along vegetative Mts. No KLP localization is seen in pollen tubes treated with antibody after it had been preadsorbed to the antigenic peptides. The antibody recognizes an M_r 110,000 polypeptide in extracts of tobacco pollen tubes, and a polypeptide of somewhat lower M_r inTradescantia pollen tubes. Our results show that KLP(s) related to KatAp are present in tobacco generative cells and may play roles in the organization and/or operation of the mitotic apparatus and phragmoplast.Abbreviations KLP kinesin-like protein - Mt microtubule - MA mitotic apparatus Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology     

Growth rate suppression of cultured mammalian cells enhances protein productivity

Suppression of proliferation of cells which contain stable or stabilized mRNA coded for a protein to be produced, a partial mimic of cell differentiation, was examined for enhancing protein production by cultured mammalian cells. Hybridoma 2E3 cells which were adapted to be interleukin-6 sensitively growth-suppressed accumulated the mRNA of IgG₁ which is reported stable, and IgG₁ production rate increased as a result when their growth was suppressed with interleukin-6. A myeloma cell line was similarly adapted; the obtained myeloma cells can be used as host cells for enhancing production of exogenous proteins by suppressing growth with interleukin-6. Temperature-sensitively growth-suppressible mutants of mouse mammary carcinoma FM3A were transfected with cDNA of IgM ₁ chain and cultured at nonpermissive temperature to enhance production of ₁. Addition of various growth-suppressive reagents to culture medium was studied for finding methods suitable for suppressing growth while maintaining high cell viability. Caffeine yielded the best results among these reagents. Deprivation of various growth-supporting components in culture medium was also tested; simultaneous deprivation of insulin and transferrin viably suppressed growth of hybridoma 2E3 cells, resulting in enhanced antibody productivity.Abbreviations IL6 recombinant human interleukin-6 - TGF- recombinant human TGF-₁ - X63.653-P3X63 Ag8.653 myeloma     

Characterization of a 42 degrees C-specific heat shock protein of mammalian cells

HeLa cells synthesize a particular heat shock protein that is induced only by heat shock at 42 degrees C, and not at 45 degrees C or by other stresses that induce major heat shock proteins (Hatayama et al. (1986) Biochem. Biophys. Res. Commun. 137, 957-963). We further characterized the 42 degrees C-specific protein. This protein was induced in mouse FM 3A cells as well as in human HeLa cells. In both cell lines, the protein was resolved into two spots, a basic polypeptide and an acidc one. The mRNA of the protein was induced during the incubation of these cells at 42 degrees C, and the in vitro translation product of mRNA corresponded to the basic, not to the acidic, polypeptide. During the chase period for cells that were labeled with [35S]-methionine, the basic polypeptide of the protein decreased, and the acidic one increased, indicating that the protein was synthesized as the basic polypeptide and then somehow modified to become the acidic one. The 42 degrees C-specific protein was found only in the cytosol fraction, and not in the nuclear or other particulate fractions, in both HeLa and FM 3A cells. The results suggested that the 42 degrees C-specific protein may have some function in the cytoplasm of mammalian cells during mild heat shock.     

The human chromokinesin Kid is a plus end-directed microtubule-based motor

Kid is a kinesin-like DNA-binding protein known to be involved in chromosome movement during mitosis, although its actual motor function has not been demonstrated. Here, we describe the initial characterization of Kid as a microtubule-based motor using optical trapping microscopy. A bacterially expressed fusion protein consisting of a truncated Kid fragment (amino acids 1-388 or 1-439) is indeed an active microtubule motor with an average speed of approximately 160 nm/s, and the polarity of movement is plus end directed. We could not detect processive movement of either monomeric Kid or dimerizing chimeric Kid; however, low levels of processivity (a few steps) cannot be detected with our method. These results are consistent with Kid having a role in chromosome congression in vivo, where it would be responsible for the polar ejection forces acting on the chromosome arms.     

The minus end-directed motor Kar3 is required for coupling dynamic microtubule plus ends to the cortical shmoo tip in budding yeast

The budding yeast shmoo tip is a model system for analyzing mechanisms coupling force production to microtubule plus-end polymerization/depolymerization. Dynamic plus ends of astral microtubules interact with the shmoo tip in mating yeast cells, positioning nuclei for karyogamy. We have used live-cell imaging of GFP fusions to identify proteins that couple dynamic microtubule plus ends to the shmoo tip. We find that Kar3p, a minus end-directed kinesin motor protein, is required, whereas the other cytoplasmic motors, dynein and the kinesins Kip2p and Kip3p, are not. In the absence of Kar3p, attached microtubule plus ends released from the shmoo tip when they switched to depolymerization. Furthermore, microtubules in cells expressing kar3-1, a mutant that results in rigor binding to microtubules [2], were stabilized specifically at shmoo tips. Imaging of Kar3p-GFP during mating revealed that fluorescence at the shmoo tip increased during periods of microtubule depolymerization. These data are the first to localize the activity of a minus end-directed kinesin at the plus ends of microtubules. We propose a model in which Kar3p couples depolymerizing microtubule plus ends to the cell cortex and the Bim1p-Kar9p protein complex maintains attachment during microtubule polymerization. In support of this model, analysis of Bim1p-GFP at the shmoo tip results in a localization pattern complementary to that of Kar3p-GFP.     

Electron tomography of vitreous sections from cultured mammalian cells

Cryo-electron tomography of appropriately thin, frozen-hydrated biological specimens has excellent potential for investigating the 3D macromolecular architecture of eukaryotic cells and tissues. Since cardiomyocytes are too thick to be visualised in an intact state, we grew immortalised cell line HL-1 to sub-confluency and harvested the cells by enzymatic detachment prior to hyperbaric freezing, ultramicrotomy, and tomography. We improved the efficiency of tomographic acquisition from vitreous cryosections by implementing two new features: (1) fluorescence microscopy at cryogenic temperatures to search for features of interest without expending any of the tolerable electron dose on secondary (non-imaging) tasks, and (2) the use of colloidal gold as fiducial markers. Vital fluorescent staining and subsequent cryo-fluorescence microscopy of vitreous sections were used to localise mitochondria lying in positions suitable for acquiring tilt series, taking into account section flatness, presence of contamination and proximity to grid bars. To provide a simple and robust means of aligning tomograms, we developed a universally applicable protocol for depositing colloidal gold onto vitreous sections, analogous to the method for applying quantum dots described by Masich et al. [Masich, S., Östberg, T., Norlén, L., Shupliakov, O., Daneholt, B., 2006. A procedure to deposit fiducial markers on vitreous cryo-sections for cellular tomography. J. Struct. Biol. 156, 461–468]. Tomograms of thin sections (nominal thickness 65–85 nm) of cardiac mitochondria revealed the interconnectivity of cristae and junctions with the inner mitochondrial membrane. In some cases, ATP synthases could be identified without ambiguity. These findings confirm the feasibility of investigating the structural biology of mammalian cells in three dimensions and at a resolution of 6–8 nm.     

Native gel analysis of macromolecular protein complexes in cultured mammalian cells

Native gel electrophoresis enables separation of cellular proteins in their non‐denatured state. In experiments aimed at analysing proteins in higher order or multimeric assemblies (i.e. protein complexes) it offers some advantages over rival approaches, particularly as an interface technology with mass spectrometry. Here we separated fractions from HEK293 cells by native electrophoresis in order to survey protein complexes in the cytoplasmic, nuclear and chromatin environments, finding 689 proteins distributed among 217 previously described complexes. As expected, different fractions contained distinct combinations of macromolecular complexes, with subunits of the same complex tending to co‐migrate. Exceptions to this observation could often be explained by the presence of subunits shared among different complexes. We investigated one identified complex, the Polycomb Repressor Complex 2 (PRC2), in more detail following affinity purification of the EZH2 subunit. This approach resulted in the identification of all previously reported members of PRC2. Overall, this work demonstrates that the use of native gel electrophoresis as an upstream separating step is an effective approach for analysis of the components and cellular distribution of protein complexes.     

Ribonuclease of cultured mammalian cells

KB cell ribonuclease has been purified 260-fold and the fundamental properties have been studied. Though the enzyme is concentrated in the lysosomal fraction, appreciable quantities are present in the cell sap and nuclear fractions. Comparison of the optimal temperature and pH for activity, and the heat stability of enzyme from these three fractions suggests that only one species of this enzyme exists in these cells. The enzyme behaves as an endonuclease, cleaving synthetic pyrimidine polynucleotides to smaller oligonucleotides with cyclic 2′:3′ end-groups. The final product is pyrimidine nucleoside 3′ monophosphate. Polyadenylic acid is not hydrolyzed. Of the properties examined in this study only two differences were noted between KB cell and pancreatic ribonuclease. KB cell enzyme acts optimally at pH 6 as opposed to an optimum at pH 7 to 8 for pancreatic enzyme. In addition ribonuclease from KB cells is definitely less stable to heating at 100°C than is the enzyme isolated from pancreas.     

Interaction of a kinesin-like protein with calmodulin isoforms from Arabidopsis.

In Arabidopsis and other plants there are multiple calmodulin isoforms. However, the role of these isoforms in regulating the activity of target proteins is obscure. Here, we analyzed the interaction between a kinesin-like calmodulin-binding motor protein (Reddy, A. S. N., Safadi, F., Narasimhulu, S. B., Golovkin, M., and Hu, X. (1996) J. Biol. Chem. 271, 7052-7060) and three calmodulin isoforms (calmodulin-2, -4, and -6) from Arabidopsis using different approaches. Gel mobility and fluorescence shift assays revealed that the motor binds to all calmodulin isoforms in a calcium-dependent manner. Furthermore, all calmodulin isoforms were able to activate bovine calcium/calmodulin-dependent phosphodiesterase. However, the concentration of calmodulin-2 required for half-maximal activation of phosphodiesterase is 2- and 6-fold lower compared with calmodulin-4 and -6, respectively. The dissociation constants of the motor to calmodulin-2, -4, and -6 are 12.8, 27.0, and 27.8 nM, respectively, indicating that calmodulin-2 has 2-fold higher affinity for the motor than calmodulin-4 and -6. Similar results were obtained using another assay that involves the binding of (35)S-labeled calmodulin isoforms to the motor. The binding saturation curves of the motor with calmodulin isoforms have confirmed that calmodulin-2 has 2-fold higher affinity to the motor. However, the affinity of calmodulin-4 and -6 isoforms for the motor was about the same. Based on these studies, we conclude that all calmodulin isoforms bind to the motor protein but with different affinities.