Abstract
The position of the microtubule spindle apparatus in mitosis dictates the plane of cell cleavage. This is critical in the positioning of daughter cells during tissue morphogenesis, and allows asymmetric cell divisions to maintain stem cells and create cell diversity. The spindle is positioned by pulling forces created by dynein in association with shrinking astral microtubules at the cell cortex. Studies in the C. elegans embryo have shown that asymmetry in the cortical pulling forces cause spindle movements and determine the size and position of daughter cells. We have previously identified an evolutionarily conserved protein complex that recruits dynein to the cortex and is required for spindle pulling forces. Our current goal is to understand how cortical pulling forces are regulated in time and space to achieve proper spindle positioning.
These studies combine several advanced methods and are focused on the protein complex that localizes dynein to the cell membrane. This complex consists of a G-protein alpha subunit as a membrane anchor, the GoLoco motif “bridge” proteins GPR-1/2 (LGN), and the dynein adaptor protein LIN-5 (NuMA). We found that spindle positioning depends on phosphorylation of LIN-5 by cell-cycle and polarity kinases, as well as nucleotide exchange of the Galpha subunit. Using CRISPR/Cas9 technology, we have created knock-in strains with specific LIN-5 phosphorylation-site mutations. To determine how these mutations affect pulling forces, we ablate the spindle midzone with a UV laser, and quantify the velocity with which the spindle poles separate. Moreover, we have tagged endogenous LIN-5 and GPR-1 with a fluorophore (eGFP), to be able to visualize the dynamics of their cortical localization in TIRF/FRAP experiments. Finally, we are exploring the use of inducible protein-protein interactions. This will enable us to leave out complex components or protein domains and study the effect on (the asymmetry of) pulling forces and complex dynamics without disrupting the entire complex.
These studies combine several advanced methods and are focused on the protein complex that localizes dynein to the cell membrane. This complex consists of a G-protein alpha subunit as a membrane anchor, the GoLoco motif “bridge” proteins GPR-1/2 (LGN), and the dynein adaptor protein LIN-5 (NuMA). We found that spindle positioning depends on phosphorylation of LIN-5 by cell-cycle and polarity kinases, as well as nucleotide exchange of the Galpha subunit. Using CRISPR/Cas9 technology, we have created knock-in strains with specific LIN-5 phosphorylation-site mutations. To determine how these mutations affect pulling forces, we ablate the spindle midzone with a UV laser, and quantify the velocity with which the spindle poles separate. Moreover, we have tagged endogenous LIN-5 and GPR-1 with a fluorophore (eGFP), to be able to visualize the dynamics of their cortical localization in TIRF/FRAP experiments. Finally, we are exploring the use of inducible protein-protein interactions. This will enable us to leave out complex components or protein domains and study the effect on (the asymmetry of) pulling forces and complex dynamics without disrupting the entire complex.
| Original language | English |
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| Publication status | Published - 29 Sept 2014 |
| Event | Dutch Biophysics meeting 2014 - , Netherlands Duration: 29 Sept 2014 → … |
Conference
| Conference | Dutch Biophysics meeting 2014 |
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| Country/Territory | Netherlands |
| Period | 29/09/14 → … |