Cytoskeleton, Cell Division, Cancer Biology
Cytoskeleton, cell division and cancer biology
We are deeply interested in the cytoskeleton, cell division and cancer biology and how chemistry can be used to unravel the underlying mechanisms. Our work has helped answer:
(i) How are proper chromosome-microtubule attachments established? We focused on key kinases (e.g. Aurora B) and phosphatases (e.g. PP2A). One important finding, which revised a long-standing model, was that chromosomes can congress to the metaphase plate without attaching to the opposite spindle poles. Our work also revealed that a spatial Aurora kinase-dependent phosphorylation gradient is established during anaphase and can coordinate the final steps of cell division.
(ii) How do functional outputs of nanometer-sized proteins scale with micrometer-sized dynamic features (e.g. microtubule length)? Two important findings are: (a) We showed how purified PRC1 and kinesin-4, which form a complex, generate micron-sized tags at microtubule plus-ends. Remarkably, the length of these tags is proportional to microtubule length in vitro and in dividing cells. (b) Our studies revealed that kinesin-5, which is required for cell division and is an anti-cancer drug target, can generate forces that scale with microtubule overlap length.
(iii) How does the metaphase spindle generate forces during division? We measured, for the first time, the orientation- and timescale-dependent micromechanical properties of the metaphase spindle. Our findings help explain how this essential structure transmits forces and how it accommodates proportionately large deformations. In addition, we focused on microtubules crosslinked by purified motor and non-motor proteins required for cell division. For non-motor proteins we show that friction associated with motion along a microtubule depends not only on velocity, but also the direction of motion. This asymmetry in friction predicted, and our experiments confirmed, that passive crosslinking proteins can undergo directional motion in active filament networks.
Current projects:
To decipher mechanisms of microtubule nucleation we are focusing on:
(i) γ-tubulin ring complex, a large multi-protein complex that is an essential regulator of microtubule formation. We reported the first cryo-EM reconstruction (~3.8A) of this complex, which revealed an unusual asymmetric cone-shaped architecture. In addition, we have developed an approach to generate this multi-protein complex in recombinant form, allowing us to dissect the contributions of key structural features and components in controlled in vitro experiments. We are building on these studies to analyze the function on this complex.
(ii) Human tubulin, the building block of microtubules. We have reported methods to generate recombinant forms of affinity-tag free recombinant human tubulin and analyzed the structures of a disease-associated mutant and different isotypes. We are examining how microtubule structural and functional diversity can result from small differences in the sequence of tubulin, a highly conserved protein.
(iii) Augmin, a hetero-octameric complex involved in microtubule-dependent microtubule formation. We are characterizing recombinant forms of the holo-complex as well as stable sub-complexes.
Recent publications:
1. Wieczorek, M., Urnavicius, L., Ti, S-C., Molloy, K.R., Chait, B.T., Kapoor, T.M. (2020), Asymetric molecular architecture of the human g-tubulin ring complex. Cell, 180, 165-75. PMC7027161.
2. Wieczorek, M., Ti, S-C., Urnavicius, L., Molloy, K.R., Aher, A., Chait, B.T., Kapoor, T.M. (2021) Biochemical reconstitutions reveal principles of human g-TuRC assembly and function. Journal of Cell Biology, 220, e202009146. PMC7844428.
Our work on cell division is focusing on:
(i) Assembly and function of the spindle midzone. To examine how the spindle midzone, an antiparallel microtubule array that emerges during anaphase, assembles and how it contributes to chromosome segregation we are combining cell and chemical biology methods with lattice-light sheet microscopy.
(iv) Diffusivity in the metaphase cytoplasm. To gain insight into the motion of mesoscale macromolecules across the mitotic cytoplasm, we are using cells expressing genetically-encoded multimeric nanoparticles (GEMs). Imaging GEMs on millisecond timescales, along with chemical probe treatments, indicated that microtubule polymerization can enhance mesoscale mobility across the metaphase cytoplasm, a region crowded with thousands of densely packed microtubules.
Recent publications:
1. Pamula, M.C., Carlini, L., Forth, S., Verma, P., Suresh, S., Legant, W.R., Khodjakov, A., Betzig, E., Kapoor, T.M. (2019), High-resolution imaging reveals how the spindle midzone impacts chromosome movement. Journal of Cell Biology, 218, 2529-44. PMC6683753.
2. Carlini, L., Brittingham, G.P., Holt, L.J., Kapoor, T.M. (2020), Microtubules enhance mesoscale effective diffusivity in the crowded metaphase cytoplasm. Developmental Cell, 54, 574-82. PMC7685229.
Learn more about our work on the chemical and structural biology of mechanoenzymes