Biology subjects

Dickey, A. M.

Publications and source records attributed to Dickey, A. M..

4 recordsLinked to original sources

bioRxiv2022

Structure of LRRK1 and mechanisms of autoinhibition and activation

Leucine Rich Repeat Kinase 1 and 2 (LRRK1 and LRRK2) are homologs in the ROCO family of proteins in humans. Despite their shared domain architecture and involvement in intracellular trafficking, their disease associations are strikingly different: LRRK2 is involved in familial Parkinsons Disease (PD) while LRRK1 is linked to bone diseases. Furthermore, PD-linked mutations in LRRK2 are typically autosomal dominant gain-of-function while those in LRRK1 are autosomal recessive loss-of-function. To understand these differences, we solved cryo-EM structures of LRRK1 in its monomeric and dimeric forms. Both differ from the corresponding LRRK2 structures. Unlike LRRK2, which is sterically autoinhibited as a monomer, LRRK1 is sterically autoinhibited in a dimer-dependent manner. LRRK1 has an additional level of autoinhibition that prevents activation of the kinase and is absent in LRRK2. Finally, we place the structural signatures of LRRK1 and LRRK2 in the context of the evolution of the LRRK family of proteins.

biochemistry↗

bioRxiv2022

Structural basis for Parkinson's Disease-linked LRRK2's binding to microtubules

Leucine Rich Repeat Kinase 2 (LRRK2) is one of the most commonly mutated genes in familial Parkinsons Disease (PD). Under some circumstances, LRRK2 co-localizes with microtubules in cells, an association enhanced by PD mutations. We report a cryo-electron microscopy structure of the catalytic half of LRRK2, containing its kinase, which is in a closed conformation, and GTPase domains, bound to microtubules. We also report a structure of the catalytic half of LRRK1, which is closely related to LRRK2, but is not linked to PD. LRRK1s structure is similar to LRRK2, but LRRK1 does not interact with microtubules. Guided by these structures, we identify amino acids in LRRK2s GTPase domain that mediate microtubule binding; mutating them disrupts microtubule binding in vitro and in cells, without affecting LRRK2s kinase activity. Our results have implications for the design of therapeutic LRRK2 kinase inhibitors.

cell biology↗

bioRxiv2018

Structurally distinct endocytic pathways for B cell receptors in B lymphocytes

B lymphocytes play a critical role in adaptive immunity. Upon antigen binding, B cell receptors (BCR) cluster on the plasma membrane and are internalized by endocytosis. In this process, B cells capture diverse antigens in various contexts and concentrations. However, it is unclear whether the mechanism of BCR endocytosis changes in response to these factors. Here, we studied the mechanism of soluble antigen-induced BCR clustering and internalization in a cultured human B cell line using correlative super resolution fluorescence and platinum replica electron microscopy. First, by visualizing nanoscale BCR clusters, we provide direct evidence that BCR cluster size increases with F(ab)2 concentration. Next, we show that the physical mechanism of internalization switches in response to BCR cluster size. At low concentrations of antigen, B cells internalize small BCR clusters by classical clathrin-mediated endocytosis. At high antigen concentrations, when clusters size increases beyond the size of a single clathrin coated pit, B cells retrieve receptor clusters using large invaginations of the plasma membrane capped with clathrin. At these sites, we observed early and sustained recruitment of actin and an actin polymerizing protein FCHSD2. We further show that actin recruitment is required for the efficient generation of these novel endocytic carriers and for their capture into the cytosol. We propose that in B cells, the mechanism of endocytosis switches to accommodate large receptor clusters formed when cells encounter high concentrations of soluble antigen. This mechanism is regulated by the organization and dynamics of the cortical actin cytoskeleton.

cell biology↗

bioRxiv2017

Membrane bending begins at any stage of clathrin-coat assembly and defines endocytic dynamics

Summary ParagraphClathrin-mediated endocytosis internalizes membrane from the cell surface by reshaping flat regions of membrane into spherical vesicles(1, 2). The relationship between membrane bending and clathrin coatomer assembly has been inferred from electron microscopy and structural biology, without directly visualization of membrane bending dynamics (3-6). This has resulted in two distinct and opposing models for how clathrin bends membrane (7-10). Here, polarized Total Internal Reflection Fluorescence microscopy was improved and combined with electron microscopy, atomic force microscopy, and super-resolution imaging to measure membrane bending during endogenous clathrin and dynamin assembly in living cells. Surprisingly, and not predicted by either model, the timing of membrane bending was variable relative to clathrin assembly. Approximately half of the time, membrane bending occurs at the start of clathrin assembly, in the other half, the onset of membrane bending lags clathrin arrival, and occasionally completely assembled flat clathrin transitions into a pit. Importantly, once the membrane bends, the process proceeds to scission with similar timing. We conclude that the pathway of coatomer formation is versatile and can bend the membrane during or after the assembly of the clathrin lattice. These results highlight the heterogeneity in this fundamental biological process, and provide a more complete nanoscale view of membrane bending dynamics during endocytosis.

cell biology↗