Biology subjects

Hazelbaker, M.

Publications and source records attributed to Hazelbaker, M..

3 recordsLinked to original sources

bioRxiv2026

Arp2/3-mediated turnover of large clathrin lattices is regulated through the tyrosine kinase ACK

Clathrin mediated endocytosis (CME) is a vital cellular process that mediates cell signaling by controlling the internalization of extracellular cargo and activated receptors. Arp2/3-branched actin provides force to assist in membrane invagination and scission in CME. Loss of Arp2/3-branched actin in conditional Arpc2 KO fibroblasts results in an increase of large arrested clathrin lattices (LACLs) visualized by live-cell TIRF imaging. Additional structural details of LACLs were revealed using electron microscopy and include an increase in arrested clathrin lattices of various curvatures. Numerous CME proteins have heightened levels of tyrosine phosphorylation at these LACLs in Arpc2 KO cells. We identified the non-receptor tyrosine kinase ACK (Activated Cdc42-Associated Kinase) as a key upstream regulator of LACL turnover. CRISPR KO of ACK abrogates LACL tyrosine phosphorylation and impairs cells capacity to resolve LACLs. Our results support a model where ACK recruitment and activation at LACLs drives branched actin formation to help resolve large accumulations of arrested CME structures.

Cell Biology↗

bioRxiv2025

SureCLIP and DrCLIP: Genetic manipulation-free, generalizable interactome mapping for cells and zebrafish

Unbiased interactome mapping currently relies on two types of approaches: co-immunoprecipitation coupled with mass spectrometry (co-IP/MS) and proximity-based tagging. Although co-IP/MS does not require genetic manipulation - thereby avoiding artifacts from exogenous protein expression and the time needed to generate stable cell or animal lines - it is increasingly being supplanted by proximity labeling techniques due to lower sensitivity and limited robustness. These drawbacks largely arise from the non-covalent nature of protein-protein interactions (PPIs) and carryover of bait and immunoglobulin (IgG) proteins in the final eluate. Here, we present a reversible crosslinker-mediated co-IP/MS workflow for endogenous proteins that (1) obviates the need for extensive lysis, wash, and elution optimization, (2) minimizes bait and IgG co-elution, and (3) captures several hundreds of interactors. We first describe subcellular-resolution, reversible crosslinker-mediated co-immunoprecipitation (SureCLIP), which enables PPI mapping of Hic-5, a focal adhesion protein, and lamin B1, a nuclear envelope protein, in cultured cells. We then introduce DrCLIP, a Danio rerio adaptation of SureCLIP, which allows comprehensive mapping of PPIs in zebrafish larvae. Using this approach, we profiled the kindlin-2 interactome in wild-type embryos without genetic manipulation. Both SureCLIP and DrCLIP exhibit high sensitivity and enable identification of novel interactors, providing a generalizable and powerful platform for defining native interactomes in 2D culture and whole animals.

cell biology↗

bioRxiv2020

Spatial Regulation of MCAK Promotes Cell Polarization and Focal Adhesion Turnover to Drive Robust Cell Migration

The asymmetric distribution of microtubule (MT) dynamics in migrating cells is important for cell polarization, yet the underlying regulatory mechanisms remain underexplored. Here, we addressed this question by studying the role of the MT depolymerase, MCAK, in the highly persistent migration of RPE-1 cells. MCAK knockdown leads to slowed migration and poor directional movement. Fixed and live cell imaging revealed that MCAK knockdown results in excessive membrane ruffling as well as defects in cell polarization and the maintenance of a major protrusive front. Additionally, loss of MCAK increases the lifetime of focal adhesions by decreasing their disassembly rate. These defects are due in part to the loss of the spatial distribution of MCAK activity, wherein activity is higher in the trailing edge of cells compared to the leading edge. Overexpression of Rac1 has a dominant effect over MCAK activity, placing it downstream or in a parallel pathway to MCAK function in migration. Together, our data support a model that places MCAK at a key nexus of a feedback loop, in which polarized distribution of MCAK activity and subsequent differential regulation of MT dynamics contributes to cell polarity and directional migration.

cell biology↗