Russell Essner

Characterization of Cep135, a novel coiled-coil centrosomal protein involved in microtubule organization in mammalian cells

By using monoclonal antibodies raised against isolated clam centrosomes, we have identified a novel 135-kD centrosomal protein (Cep135), present in a wide range of organisms. Cep135 is located at the centrosome throughout the cell cycle, and localization is independent of the microtubule network. It distributes throughout the centrosomal area in association with the electron-dense material surrounding centrioles. Sequence analysis of cDNA isolated from CHO cells predicted a protein of 1,145–amino acid residues with extensive α-helical domains. Expression of a series of deletion constructs revealed the presence of three independent centrosome-targeting domains. Overexpression of Cep135 resulted in the accumulation of unique whorl-like particles in both the centrosome and the cytoplasm. Although their size, shape, and number varied according to the level of protein expression, these whorls were composed of parallel dense lines arranged in a 6-nm space. Altered levels of Cep135 by protein overexpression and/or suppression of endogenous Cep135 by RNA interference caused disorganization of interphase and mitotic spindle microtubules. Thus, Cep135 may play an important role in the centrosomal function of organizing microtubules in mammalian cells.

Filamentous polymers induced by overexpression of a novel centrosomal protein, Cep135

A novel 135 kDa centrosomal component (Cep135) was identified by immunoscreening of a mammalian expression library with monoclonal antibodies raised against clam centrosomes. It is predicted to be a highly coiled‐coil protein with an extensive α‐helix, suggesting that Cep135 is a structural component of the centrosome. To evaluate how the protein is arranged in the centrosomal structure, we overexpressed Cep135 polypeptides in CHO cells by transient transfection. HA‐ or GFP‐tagged full (amino acids 1–1144) as well as truncated (#10, 29–1144; Δ3, 29–812) polypeptides become localized at the centrosome and induce cytoplasmic dots of various size and number in CHO cells. Centrosomes are associated with massive ∼7 nm filaments and dense particles organized in a whorl‐like arrangement in which parallel‐oriented dense lines appear with a regular ∼7 nm periodicity. The same filamentous aggregates are also detected in cytoplasmic dots, indicating that overexpressed Cep135 can assemble into elaborate higher‐ordered structures in and outside the centrosome. Sf9 cells infected with baculovirus containing Cep135 sequences induce filamentous polymers which are distinctive from the whorl seen in CHO cells; #10 forms highly packed spheroids, but the Δ3‐containing structure looks loose. Both structures show an internal repeating unit of dense and less dense stripes. Although the distance between the outer end of two adjacent dense lines is similar between two types of polymers (∼120 nm), the dense stripe of Δ3 polymers (∼40 nm) is wider than #10 (∼30 nm). The light band of Δ3 (∼40 nm) is thus narrower than #10 (∼60 nm). Since thin fibers are frequently seen to extend from one dense line to the next, the coiled‐coil rod of Cep135 may span the light band. These results suggest that overexpressed Cep135 assemble into distinctive polymers in a domain‐specific manner. Microsc. Res. Tech. 49:478–486, 2000.

Cell cycle-dependent expression of the CHO2 antigen, a minus-end directed kinesin-like motor in mammalian cells

SummaryA 69 kDa protein present in the interphase centrosome and mitotic spindle/pole was identified with the CHO2 monoclonal antibodies raised against mitotic spindles isolated from Chinese hamster ovary (CHO) cells. Isolation and characterization of antigen-specific cDNAs and recombinant proteins demonstrated that the protein is a minus-end-directed microtubule motor with the motor domain located at the C terminus. Affinity-purified polyclonal antibodies prepared to bacterially expressed fusion proteins revealed the presence of the antigen in interphase nuclei, and the degree of nuclear immunostaining intensity varied among cells at different cell cycle stages. In order to examine the change of antigen expression during the cell cycle, we prepared synchronized populations of CHO cells, double stained with CHO2 and PCNA (proliferating cell nuclear antigen) antibodies, and quantitated the amounts of nuclear fluorescence using the MetaMorpho image analysis software package. Cells right after cell division contained nuclei with the lowest level of CHO2 immunofluorescence. The immunofluorescence intensity progressively increases through G1 to S, reaching a maximum level by the end of G2. The antibody uniformly stained the entire nuclear region, and the total amount of fluorescence detected in G2 cells was greater than three times that of G1 cells. Cell cycle dependent accumulation of the CHO2 antigen was further confirmed by immunoblot analysis of the protein included in whole cell extracts and nuclei isolated from synchronized CHO cells. Northern blot analysis showed that, although the CHO2-transcript accumulated during later stages of the cell cycle, its abundance declined through G1 to S, and was lowest in cells at the early S phase. The difference in the expression pattern of the antigen protein and its transcript may suggest the presence of multiple mechanisms controlling the level of CHO2 antigen during the course of the cell cycle.

Interaction of an overexpressed γ-tubulin with microtubules in vivo and in vitro

Abstract γ-Tubulin is an ubiquitous MTOC (microtubule-organizing center) component essential for the regulation of microtubule functions. A 1.8 kb cDNA coding for γ-tubulin was isolated from CHO cells. Analysis of nucleotide sequence predicts a protein of 451 amino acids, which is over 97% identical to human and Xenopus γ-tubulin. When CHO cells were transiently transfected with the γ-tubulin clone, epitope-tagged full-length, as well as truncated polypeptides (amino acids 1-398 and 1-340), resulted in the formation of cytoplasmic foci of various sizes. Although one of the foci was identified as the centrosome, the rest of the dots were not associated with any other centrosomal components tested so far. The pattern of microtubule organization was not affected by induction of such γ-tubulin-containing dots in transfected cells. In addition, the cytoplasmic foci were unable to serve as the site for microtubule regrowth in nocodazole-treated cells upon removal of the drug, suggesting that γ-tubulin-containing foci were not involved in the activity for microtubule formation and organization. Using the monomeric form of Chlamydomonas γ-tubulin purified from insect Sf9 cells (Vassilev et al., J. Cell Sci. 108: 1083, 1995), interaction between γ-tubulin and microtubules was further investigated by immunoelectron microscopy. Microtubules incubated with γ-tubulin monomers in vitro were associated with more gold particles conjugated with γ-tubulin than in controls where no exogenous γ-tubulin was added. However, binding of γ-tubulin to microtubules was not extensive and was easily lost during sample preparation. Although γ-tubulin was detected at the minus end of microtubules several times more frequently than the plus end, the majority of gold particles were seen along the microtubule length. These results contradict the previous reports (Li and Joshi, J. Cell Biol. 131: 207, 1995; Shu and Joshi, J. Cell Biol. 130: 1137, 1995), which might be ascribed to the difference in the level of protein expression in transfected cells.Abstract γ-Tubulin is an ubiquitous MTOC (microtubule-organizing center) component essential for the regulation of microtubule functions. A 1.8 kb cDNA coding for γ-tubulin was isolated from CHO cells. Analysis of nucleotide sequence predicts a protein of 451 amino acids, which is over 97% identical to human and Xenopus γ-tubulin. When CHO cells were transiently transfected with the γ-tubulin clone, epitope-tagged full-length, as well as truncated polypeptides (amino acids 1-398 and 1-340), resulted in the formation of cytoplasmic foci of various sizes. Although one of the foci was identified as the centrosome, the rest of the dots were not associated with any other centrosomal components tested so far. The pattern of microtubule organization was not affected by induction of such γ-tubulin-containing dots in transfected cells. In addition, the cytoplasmic foci were unable to serve as the site for microtubule regrowth in nocodazole-treated cells upon removal of the drug, suggesting that γ-tubulin-containing foci were not involved in the activity for microtubule formation and organization. Using the monomeric form of Chlamydomonas γ-tubulin purified from insect Sf9 cells (Vassilev et al., J. Cell Sci. 108: 1083, 1995), interaction between γ-tubulin and microtubules was further investigated by immunoelectron microscopy. Microtubules incubated with γ-tubulin monomers in vitro were associated with more gold particles conjugated with γ-tubulin than in controls where no exogenous γ-tubulin was added. However, binding of γ-tubulin to microtubules was not extensive and was easily lost during sample preparation. Although γ-tubulin was detected at the minus end of microtubules several times more frequently than the plus end, the majority of gold particles were seen along the microtubule length. These results contradict the previous reports (Li and Joshi, J. Cell Biol. 131: 207, 1995; Shu and Joshi, J. Cell Biol. 130: 1137, 1995), which might be ascribed to the difference in the level of protein expression in transfected cells.

Interaction of an Overexpressed γ-Tubulin with MicrotubulesIn VivoandIn Vitro

Abstract γ-Tubulin is an ubiquitous MTOC (microtubule-organizing center) component essential for the regulation of microtubule functions. A 1.8 kb cDNA coding for γ-tubulin was isolated from CHO cells. Analysis of nucleotide sequence predicts a protein of 451 amino acids, which is over 97% identical to human and Xenopus γ-tubulin. When CHO cells were transiently transfected with the γ-tubulin clone, epitope-tagged full-length, as well as truncated polypeptides (amino acids 1-398 and 1-340), resulted in the formation of cytoplasmic foci of various sizes. Although one of the foci was identified as the centrosome, the rest of the dots were not associated with any other centrosomal components tested so far. The pattern of microtubule organization was not affected by induction of such γ-tubulin-containing dots in transfected cells. In addition, the cytoplasmic foci were unable to serve as the site for microtubule regrowth in nocodazole-treated cells upon removal of the drug, suggesting that γ-tubulin-containing foci were not involved in the activity for microtubule formation and organization. Using the monomeric form of Chlamydomonas γ-tubulin purified from insect Sf9 cells (Vassilev et al., J. Cell Sci. 108: 1083, 1995), interaction between γ-tubulin and microtubules was further investigated by immunoelectron microscopy. Microtubules incubated with γ-tubulin monomers in vitro were associated with more gold particles conjugated with γ-tubulin than in controls where no exogenous γ-tubulin was added. However, binding of γ-tubulin to microtubules was not extensive and was easily lost during sample preparation. Although γ-tubulin was detected at the minus end of microtubules several times more frequently than the plus end, the majority of gold particles were seen along the microtubule length. These results contradict the previous reports (Li and Joshi, J. Cell Biol. 131: 207, 1995; Shu and Joshi, J. Cell Biol. 130: 1137, 1995), which might be ascribed to the difference in the level of protein expression in transfected cells.Abstract γ-Tubulin is an ubiquitous MTOC (microtubule-organizing center) component essential for the regulation of microtubule functions. A 1.8 kb cDNA coding for γ-tubulin was isolated from CHO cells. Analysis of nucleotide sequence predicts a protein of 451 amino acids, which is over 97% identical to human and Xenopus γ-tubulin. When CHO cells were transiently transfected with the γ-tubulin clone, epitope-tagged full-length, as well as truncated polypeptides (amino acids 1-398 and 1-340), resulted in the formation of cytoplasmic foci of various sizes. Although one of the foci was identified as the centrosome, the rest of the dots were not associated with any other centrosomal components tested so far. The pattern of microtubule organization was not affected by induction of such γ-tubulin-containing dots in transfected cells. In addition, the cytoplasmic foci were unable to serve as the site for microtubule regrowth in nocodazole-treated cells upon removal of the drug, suggesting that γ-tubulin-containing foci were not involved in the activity for microtubule formation and organization. Using the monomeric form of Chlamydomonas γ-tubulin purified from insect Sf9 cells (Vassilev et al., J. Cell Sci. 108: 1083, 1995), interaction between γ-tubulin and microtubules was further investigated by immunoelectron microscopy. Microtubules incubated with γ-tubulin monomers in vitro were associated with more gold particles conjugated with γ-tubulin than in controls where no exogenous γ-tubulin was added. However, binding of γ-tubulin to microtubules was not extensive and was easily lost during sample preparation. Although γ-tubulin was detected at the minus end of microtubules several times more frequently than the plus end, the majority of gold particles were seen along the microtubule length. These results contradict the previous reports (Li and Joshi, J. Cell Biol. 131: 207, 1995; Shu and Joshi, J. Cell Biol. 130: 1137, 1995), which might be ascribed to the difference in the level of protein expression in transfected cells.

Interaction of an overexpressed gamma-tubulin with microtubules

Abstract γ-Tubulin is an ubiquitous MTOC (microtubule-organizing center) component essential for the regulation of microtubule functions. A 1.8 kb cDNA coding for γ-tubulin was isolated from CHO cells. Analysis of nucleotide sequence predicts a protein of 451 amino acids, which is over 97% identical to human and Xenopus γ-tubulin. When CHO cells were transiently transfected with the γ-tubulin clone, epitope-tagged full-length, as well as truncated polypeptides (amino acids 1-398 and 1-340), resulted in the formation of cytoplasmic foci of various sizes. Although one of the foci was identified as the centrosome, the rest of the dots were not associated with any other centrosomal components tested so far. The pattern of microtubule organization was not affected by induction of such γ-tubulin-containing dots in transfected cells. In addition, the cytoplasmic foci were unable to serve as the site for microtubule regrowth in nocodazole-treated cells upon removal of the drug, suggesting that γ-tubulin-containing foci were not involved in the activity for microtubule formation and organization. Using the monomeric form of Chlamydomonas γ-tubulin purified from insect Sf9 cells (Vassilev et al., J. Cell Sci. 108: 1083, 1995), interaction between γ-tubulin and microtubules was further investigated by immunoelectron microscopy. Microtubules incubated with γ-tubulin monomers in vitro were associated with more gold particles conjugated with γ-tubulin than in controls where no exogenous γ-tubulin was added. However, binding of γ-tubulin to microtubules was not extensive and was easily lost during sample preparation. Although γ-tubulin was detected at the minus end of microtubules several times more frequently than the plus end, the majority of gold particles were seen along the microtubule length. These results contradict the previous reports (Li and Joshi, J. Cell Biol. 131: 207, 1995; Shu and Joshi, J. Cell Biol. 130: 1137, 1995), which might be ascribed to the difference in the level of protein expression in transfected cells.Abstract γ-Tubulin is an ubiquitous MTOC (microtubule-organizing center) component essential for the regulation of microtubule functions. A 1.8 kb cDNA coding for γ-tubulin was isolated from CHO cells. Analysis of nucleotide sequence predicts a protein of 451 amino acids, which is over 97% identical to human and Xenopus γ-tubulin. When CHO cells were transiently transfected with the γ-tubulin clone, epitope-tagged full-length, as well as truncated polypeptides (amino acids 1-398 and 1-340), resulted in the formation of cytoplasmic foci of various sizes. Although one of the foci was identified as the centrosome, the rest of the dots were not associated with any other centrosomal components tested so far. The pattern of microtubule organization was not affected by induction of such γ-tubulin-containing dots in transfected cells. In addition, the cytoplasmic foci were unable to serve as the site for microtubule regrowth in nocodazole-treated cells upon removal of the drug, suggesting that γ-tubulin-containing foci were not involved in the activity for microtubule formation and organization. Using the monomeric form of Chlamydomonas γ-tubulin purified from insect Sf9 cells (Vassilev et al., J. Cell Sci. 108: 1083, 1995), interaction between γ-tubulin and microtubules was further investigated by immunoelectron microscopy. Microtubules incubated with γ-tubulin monomers in vitro were associated with more gold particles conjugated with γ-tubulin than in controls where no exogenous γ-tubulin was added. However, binding of γ-tubulin to microtubules was not extensive and was easily lost during sample preparation. Although γ-tubulin was detected at the minus end of microtubules several times more frequently than the plus end, the majority of gold particles were seen along the microtubule length. These results contradict the previous reports (Li and Joshi, J. Cell Biol. 131: 207, 1995; Shu and Joshi, J. Cell Biol. 130: 1137, 1995), which might be ascribed to the difference in the level of protein expression in transfected cells.