Blutrich, R. | bioRxiv Science

bioRxiv2024

Microtubule plus-end dynamics are tightly coupled to the turnover of the MOR1 polymerase

MOR1/XMAP215/Dis1/Stu2 family proteins function as polymerases that promote microtubule assembly through tubulin-interacting N-terminal tumor-overexpressed gene (TOG) domains. It remains unclear how the polymerase activity of these proteins is coordinated with their movement along the growing microtubule lattice. Previously, it was found that a L174F mutation in the N-terminal TOG domain of the 217 kDa Arabidopsis thaliana MICROTUBULE ORGANIZATION 1 (MOR1) protein impaired microtubule growth and shrinkage rates in a temperature-dependent manner. In this study, we used a homologous recombination strategy to generate stable transgenic lines expressing full-length MOR1 and mutant mor1-1L174F proteins tagged with the yellow fluorescent protein YPet. When expressed in mor1 mutant backgrounds, MOR1-3xYPet showed strong association with both the growing and shrinking microtubule plus ends, consistent with its involvement in both polymerization and depolymerization. By contrast, when expressed in a wild-type background, MOR1-3xYPet was redistributed along the microtubule lattice, microtubule growth rates were reduced, and plant growth was stunted. By quantifying fluorescence recovery after photobleaching, we confirmed that MOR1 has greater affinity for the plus end than the lattice. Exploiting the FRAP technique further, we identified a positive and linear correlation between microtubule polymerization rates and MOR1 turnover at the microtubule plus end. When microtubule growth rates were reduced in the mor1-1-YPet line by shifting the temperature from 21 to 30{degrees}C and for MOR1-3xYPet under taxol treatments, MOR1s turnover was delayed. In contrast, microtubule growth rates and MOR1 turnover were hastened at 30{degrees}C or after CRISPR/Cas9-mediated knock out of the CLASP protein. These findings support a new model whereby MOR1 has finite processivity on the microtubule, catalyzing a limited fixed number of tubulin additions before dissociation. HIGHLIGHTSMOR1 has high affinity for both growing and shrinking microtubule plus ends, consistent with a function in both polymerization and depolymerization events. MOR1 has greater affinity for microtubule plus ends than the lattice, and lattice binding associated with overexpression is detrimental to plant growth. MOR1 transiently associates with the microtubule plus end, the duration of which is tightly coupled with its polymerase activity.

cell biology↗

bioRxiv2021

Large cells activate global protein degradation to maintain cell size homeostasis

Proliferating animal cells maintain a stable size distribution over generations despite fluctuations in cell growth and division size. This tight control of cell size involves both cell size checkpoints, which delay cell cycle progression in small cells, and size-dependent regulation of mass accumulation rates. While we previously identified the p38 MAPK pathway as a key regulator of the mammalian cell size checkpoint, the mechanism of size-dependent growth rate regulation has remained elusive. Here, we quantified global rates of protein synthesis and degradation in cells of varying sizes, both under unperturbed conditions and in response to perturbations that trigger size-dependent compensatory growth slowdown. We found that protein synthesis rates scale proportionally with cell size across cell cycle stages and experimental conditions. In contrast, oversized cells that undergo compensatory growth slowdown exhibit a superlinear increase in proteasome-mediated protein degradation, with accelerated protein turnover per unit mass, suggesting activation of the proteasomal degradation pathway. Both nascent and long-lived proteins contribute to the elevated protein degradation during compensatory growth slowdown, with long-lived proteins playing a crucial role at the G1/S transition. Notably, large G1/S cells exhibit particularly high efficiency in protein degradation, surpassing that of similarly sized or larger cells in S and G2, coinciding with the timing of the most stringent size control in animal cells. These results collectively suggest that oversized cells reduce their growth efficiency by activating global proteasome-mediated protein degradation to promote cell size homeostasis.

cell biology↗