Brinkert, P. | bioRxiv Science

bioRxiv2021

Endocytic vacuole formation by WASH-mediated endocytosis

Endocytosis is a fundamental cellular process facilitated by diverse mechanisms. Remarkably, several distinct clathrin-independent endocytic processes were identified and characterized following virus uptake into cells. For some, however, mechanistic execution and biological function remain largely unclear. This includes an endocytic process exploited by human papillomavirus type 16 (HPV16). Using HPV16, we examined how vesicles are formed by combining systematic cellular perturbations with electron and video microscopy. Cargo uptake occurred by uncoated, inward-budding pits. Mechanistically, vesicle scission was facilitated by actin polymerization controlled through the actin nucleation promoting factor WASH. While WASH typically functions in conjunction with the retromer complex on endosomes during retrograde trafficking, endocytic vesicle formation was largely independent of retromer itself and the heterodimeric membrane-bending SNX-BAR retromer adaptor, thereby uncovering a role of WASH in endocytosis in addition to its canonical role in intracellular membrane trafficking.

cell biology↗

bioRxiv2019

Clathrin’s adaptor interaction sites are repurposed to stabilize microtubules during mitosis

Clathrin plays an important role to ensure mitotic spindle stability and efficient chromosome alignment, independently of its well-characterized functions in vesicle trafficking. While clathrin clearly localizes to the mitotic spindle and kinetochore-fiber microtubule bundles, the mechanisms by which clathrin stabilizes microtubules have remained elusive. Here we show that clathrin adaptor interaction sites on clathrin heavy chain (CHC) are repurposed during mitosis to directly recruit the microtubule-stabilizing protein GTSE1 to the mitotic spindle. Structural analyses reveal that multiple clathrin-box motifs on GTSE1 interact directly with different clathrin adaptor interaction sites on CHC, in a manner structurally analogous to that which occurs between adaptor proteins and CHC near membranes. Specific disruption of this interaction in cells releases GTSE1 from spindles and causes defects in chromosome alignment. Surprisingly, this disruption causes destabilization of astral microtubules, but not kinetochore-microtubule attachments, and the resulting chromosome alignment defect is due to a failure of chromosome congression independent of kinetochore-microtubule attachment stability. Finally, we show that GTSE1 recruited to the spindle by clathrin stabilizes microtubules and promotes chromosome congression by inhibiting the activity of the microtubule depolymerase MCAK. This work thus uncovers a novel role of clathrin to stabilize non-kinetochore-fiber microtubules to support chromosome congression. This role is carried out via clathrin adaptor-type interactions of CHC with GTSE1, defining for the first time an important repurposing of this endocytic interaction mechanism during mitosis.

cell biology↗