Spatially controlled microtubule nucleation and organization from crosslinker MAP65 condensates

Abstract

Microtubule organization in cells is essential for the internal structure and coordination of events of intracellular transport, mitosis, and cell motility. For many cell types, microtubule organization is dominated by centrosomal nucleation that use gamma-tubulin to template filaments. Yet, some cell types lack centrosomes or centrioles, such as plant cells. Instead, microtubules nucleate from regions with high concentrations of microtubule binding and nucleating proteins. A mechanism that can drive high local concentrations of nucleators is liquid-liquid phase separation of proteins with intrinsically disordered regions. Here, we report that the plant microtubule nucleator and crosslinking protein, MAP65-1, can form phase separated condensates at physiological salt and temperature without extra crowding agents. These condensates are liquid at first and can mature to gel-like phases over time and with different environmental conditions. We show that these condensates can nucleate and grow microtubule bundles that form asters, regardless of the viscoelasticity of the condensate. When gel-like droplets nucleate and grow asters from a shell of tubulin at the surface, the microtubules are able to re-fluidize the MAP65 condensate. Condensate-induced cytoskeletal formation could be a universal mechanism for organization of the microtubule and actin cytoskeletons in all cell types, especially cells without centrosomes.

Sequence-dependent fusion dynamics and physical properties of DNA droplets

“…The fluorescence intensities of the ROI and reference region were obtained using the FluoView software (version 4.0, Olympus, Tokyo, Japan). The fluorescence intensity of the ROI was normalized using the intensity of the reference region based on previously reported methods 35,36 with some modifications. The normalized intensity data were fit to the following equation: where α and β are constant terms and τ is the recovery time constant.…”

“…The normalized intensity data were fit to the following equation: where α and β are constant terms and τ is the recovery time constant. The apparent diffusion coefficient ( D apparent ) was calculated using the following equation: 35 D apparent = r 2 / τ where r is the radius of the ROI.…”

“…The frame interval was set to 1 s, and five frames were obtained before bleaching. Then, a circular region of interest (ROI) at the centre of the droplets was bleached for 1. methods 35,36 with some modifications. The normalized intensity data were fit to the following equation:…”