Limits...
Saccharomyces cerevisiae Duo1p and Dam1p, novel proteins involved in mitotic spindle function.

Hofmann C, Cheeseman IM, Goode BL, McDonald KL, Barnes G, Drubin DG - J. Cell Biol. (1998)

Bottom Line: By expressing a GFP-Dam1p fusion protein in yeast, Dam1p was also shown to be associated with intranuclear spindle microtubules and spindle pole bodies in vivo.Biochemical experiments demonstrated that Dam1p binds directly to microtubules with micromolar affinity.We suggest that Dam1p might localize Duo1p to intranuclear microtubules and spindle pole bodies to provide a previously unrecognized function (or functions) required for mitosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3202, USA.

ABSTRACT
In this paper, we describe the identification and characterization of two novel and essential mitotic spindle proteins, Duo1p and Dam1p. Duo1p was isolated because its overexpression caused defects in mitosis and a mitotic arrest. Duo1p was localized by immunofluorescence, by immunoelectron microscopy, and by tagging with green fluorescent protein (GFP), to intranuclear spindle microtubules and spindle pole bodies. Temperature-sensitive duo1 mutants arrest with short spindles. This arrest is dependent on the mitotic checkpoint. Dam1p was identified by two-hybrid analysis as a protein that binds to Duo1p. By expressing a GFP-Dam1p fusion protein in yeast, Dam1p was also shown to be associated with intranuclear spindle microtubules and spindle pole bodies in vivo. As with Duo1p, overproduction of Dam1p caused mitotic defects. Biochemical experiments demonstrated that Dam1p binds directly to microtubules with micromolar affinity. We suggest that Dam1p might localize Duo1p to intranuclear microtubules and spindle pole bodies to provide a previously unrecognized function (or functions) required for mitosis.

Show MeSH

Related in: MedlinePlus

Localization of  Dam1p. DDY759 was transformed with a plasmid expressing a galactose-inducible GFP–Dam1p fusion  protein. Cells were grown to  early log phase in minimal  medium containing glucose,  and then the cells were  washed and shifted to and  maintained in minimal medium containing 2% glycerol  for 10 h. The cells were then  washed and shifted to medium containing 2% galactose. After 5–6 h of induction, cells were fixed and  stained with an antitubulin  antibody (middle column)  and DAPI for DNA staining  (right column). The GFP fluorescence from GFP–Dam1p  (left column) colocalizes  with the intranuclear spindle  microtubules and spindle  pole bodies. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132964&req=5

Figure 5: Localization of Dam1p. DDY759 was transformed with a plasmid expressing a galactose-inducible GFP–Dam1p fusion protein. Cells were grown to early log phase in minimal medium containing glucose, and then the cells were washed and shifted to and maintained in minimal medium containing 2% glycerol for 10 h. The cells were then washed and shifted to medium containing 2% galactose. After 5–6 h of induction, cells were fixed and stained with an antitubulin antibody (middle column) and DAPI for DNA staining (right column). The GFP fluorescence from GFP–Dam1p (left column) colocalizes with the intranuclear spindle microtubules and spindle pole bodies. Bar, 10 μm.

Mentions: To determine the intracellular localization of Dam1p, a GFP–Dam1p fusion protein was expressed on a plasmid using a galactose-inducible promoter. GFP–Dam1p was found to be associated with intranuclear spindle microtubules and spindle pole bodies throughout the cell cycle, but not with cytoplasmic microtubules (Fig. 5). Similar localization has been observed by expressing an epitope-tagged Dam1p at endogenous levels in yeast (Jones, M., and M. Winey, personal communication). In this case, Dam1p was only detected at spindle pole bodies and along intranuclear microtubules of the shortest spindles observed in the culture.


Saccharomyces cerevisiae Duo1p and Dam1p, novel proteins involved in mitotic spindle function.

Hofmann C, Cheeseman IM, Goode BL, McDonald KL, Barnes G, Drubin DG - J. Cell Biol. (1998)

Localization of  Dam1p. DDY759 was transformed with a plasmid expressing a galactose-inducible GFP–Dam1p fusion  protein. Cells were grown to  early log phase in minimal  medium containing glucose,  and then the cells were  washed and shifted to and  maintained in minimal medium containing 2% glycerol  for 10 h. The cells were then  washed and shifted to medium containing 2% galactose. After 5–6 h of induction, cells were fixed and  stained with an antitubulin  antibody (middle column)  and DAPI for DNA staining  (right column). The GFP fluorescence from GFP–Dam1p  (left column) colocalizes  with the intranuclear spindle  microtubules and spindle  pole bodies. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132964&req=5

Figure 5: Localization of Dam1p. DDY759 was transformed with a plasmid expressing a galactose-inducible GFP–Dam1p fusion protein. Cells were grown to early log phase in minimal medium containing glucose, and then the cells were washed and shifted to and maintained in minimal medium containing 2% glycerol for 10 h. The cells were then washed and shifted to medium containing 2% galactose. After 5–6 h of induction, cells were fixed and stained with an antitubulin antibody (middle column) and DAPI for DNA staining (right column). The GFP fluorescence from GFP–Dam1p (left column) colocalizes with the intranuclear spindle microtubules and spindle pole bodies. Bar, 10 μm.
Mentions: To determine the intracellular localization of Dam1p, a GFP–Dam1p fusion protein was expressed on a plasmid using a galactose-inducible promoter. GFP–Dam1p was found to be associated with intranuclear spindle microtubules and spindle pole bodies throughout the cell cycle, but not with cytoplasmic microtubules (Fig. 5). Similar localization has been observed by expressing an epitope-tagged Dam1p at endogenous levels in yeast (Jones, M., and M. Winey, personal communication). In this case, Dam1p was only detected at spindle pole bodies and along intranuclear microtubules of the shortest spindles observed in the culture.

Bottom Line: By expressing a GFP-Dam1p fusion protein in yeast, Dam1p was also shown to be associated with intranuclear spindle microtubules and spindle pole bodies in vivo.Biochemical experiments demonstrated that Dam1p binds directly to microtubules with micromolar affinity.We suggest that Dam1p might localize Duo1p to intranuclear microtubules and spindle pole bodies to provide a previously unrecognized function (or functions) required for mitosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3202, USA.

ABSTRACT
In this paper, we describe the identification and characterization of two novel and essential mitotic spindle proteins, Duo1p and Dam1p. Duo1p was isolated because its overexpression caused defects in mitosis and a mitotic arrest. Duo1p was localized by immunofluorescence, by immunoelectron microscopy, and by tagging with green fluorescent protein (GFP), to intranuclear spindle microtubules and spindle pole bodies. Temperature-sensitive duo1 mutants arrest with short spindles. This arrest is dependent on the mitotic checkpoint. Dam1p was identified by two-hybrid analysis as a protein that binds to Duo1p. By expressing a GFP-Dam1p fusion protein in yeast, Dam1p was also shown to be associated with intranuclear spindle microtubules and spindle pole bodies in vivo. As with Duo1p, overproduction of Dam1p caused mitotic defects. Biochemical experiments demonstrated that Dam1p binds directly to microtubules with micromolar affinity. We suggest that Dam1p might localize Duo1p to intranuclear microtubules and spindle pole bodies to provide a previously unrecognized function (or functions) required for mitosis.

Show MeSH
Related in: MedlinePlus