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The kinesin-related proteins, Kip2p and Kip3p, function differently in nuclear migration in yeast
R K Miller1, K K Heller, L Frisèn
1Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, USA.
Abstract:
The roles of two kinesin-related proteins, Kip2p and Kip3p, in microtubule function and nuclear migration were investigated. Deletion of either gene resulted in nuclear migration defects similar to those described for dynein and kar9 mutants. By indirect immunofluorescence, the cytoplasmic microtubules in kip2Delta were consistently short or absent throughout the cell cycle. In contrast, in kip3Delta strains, the cytoplasmic microtubules were significantly longer than wild type at telophase. Furthermore, in the kip3Delta cells with nuclear positioning defects, the cytoplasmic microtubules were misoriented and failed to extend into the bud. Localization studies found Kip2p exclusively on cytoplasmic microtubules throughout the cell cycle, whereas GFP-Kip3p localized to both spindle and cytoplasmic microtubules. Genetic analysis demonstrated that the kip2Delta kar9Delta double mutants were synthetically lethal, whereas kip3Delta kar9Delta double mutants were viable. Conversely, kip3Delta dhc1Delta double mutants were synthetically lethal, whereas kip2Delta dhc1Delta double mutants were viable. We suggest that the kinesin-related proteins, Kip2p and Kip3p, function in nuclear migration and that they do so by different mechanisms. We propose that Kip2p stabilizes microtubules and is required as part of the dynein-mediated pathway in nuclear migration. Furthermore, we propose that Kip3p functions, in part, by depolymerizing microtubules and is required for the Kar9p-dependent orientation of the cytoplasmic microtubules.
Insights
Two kinesin proteins, Kip2p and Kip3p, are crucial for nuclear migration. Kip2p stabilizes microtubules in the dynein pathway, while Kip3p depolymerizes them for Kar9p-mediated orientation.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- Nuclear migration is essential for cell division and requires precise microtubule organization.
- Kinesins are motor proteins that play diverse roles in intracellular transport and cytoskeletal dynamics.
- Previous studies implicated dynein and Kar9p in nuclear positioning, but the roles of specific kinesins remained unclear.
Purpose of the Study:
- To investigate the distinct roles of kinesin-related proteins Kip2p and Kip3p in microtubule function and nuclear migration.
- To elucidate the mechanisms by which Kip2p and Kip3p contribute to the dynein- and Kar9p-dependent pathways of nuclear positioning.
Main Methods:
- Gene deletion analysis to create kip2Δ and kip3Δ mutants.
- Indirect immunofluorescence microscopy to visualize cytoplasmic microtubules.
- Fluorescent protein tagging (GFP) for protein localization studies.
- Synthetic lethality assays to analyze double mutant interactions (kip2Δ kar9Δ, kip3Δ kar9Δ, kip3Δ dhc1Δ, kip2Δ dhc1Δ).
Main Results:
- Deletion of KIP2 or KIP3 caused nuclear migration defects, similar to dynein and kar9 mutants.
- kip2Δ cells exhibited short or absent cytoplasmic microtubules; kip3Δ cells showed elongated microtubules that were misoriented and failed to bud in some cases.
- Kip2p localized to cytoplasmic microtubules, while Kip3p localized to both spindle and cytoplasmic microtubules.
- kip2Δ kar9Δ double mutants were synthetically lethal, indicating functional overlap with Kar9p pathway, whereas kip3Δ kar9Δ mutants were viable.
- kip3Δ dhc1Δ double mutants were synthetically lethal, suggesting Kip3p functions in the dynein pathway, while kip2Δ dhc1Δ mutants were viable.
Conclusions:
- Kip2p and Kip3p are essential kinesins involved in nuclear migration, operating through distinct mechanisms.
- Kip2p likely stabilizes microtubules and functions within the dynein-mediated nuclear migration pathway.
- Kip3p appears to function, in part, by depolymerizing microtubules and is crucial for Kar9p-dependent cytoplasmic microtubule orientation.