Biology subjects

Chastney, M.

Publications and source records attributed to Chastney, M..

3 recordsLinked to original sources

bioRxiv2023

Specialized actin nanoscale layers control focal adhesion turnover

Focal adhesions (FAs) connect inner workings of the cell to the extracellular matrix to control cell adhesion, migration, and mechanosensing1,2. Previous studies demonstrated that FAs contain three vertical layers, which connect extracellular matrix to the cytoskeleton3,4,5. However, cellular processes rely on precisely-regulated FA turnover, but the molecular machineries that control FA assembly and disassembly have remained elusive. By using super-resolution iPALM microscopy, we identified two unprecedented nanoscale layers within FAs, specified by actin filaments bound to tropomyosin isoforms Tpm1.6 and Tpm3.2. The Tpm1.6-actin filaments beneath the previously identified actin-regulatory layer are critical for adhesion maturation and controlled cell motility, whereas the Tpm3.2-actin filament layer towards the bottom of FA facilitates adhesion disassembly. Mechanistically, Tpm3.2 stabilizes KANK-family proteins at adhesions, and hence targets microtubule plus-ends to FAs to catalyse their disassembly. Loss of Tpm3.2 leads to disorganized microtubule network, abnormally stable FAs, and defects in tail retraction during cell migration. Thus, FAs are composed of at least three distinct actin filament layers, each having specific roles in coupling of adhesion to the cytoskeleton, or in controlling adhesion dynamics. In a broader context, these findings demonstrate how distinct actin filament populations can co-exist and perform specific functions within a defined cellular compartment.

cell biology↗

bioRxiv2022

Regulation of cell dynamics by rapid transport of integrins through the biosynthetic pathway

Cells sense and respond to the extracellular matrix (ECM) milieu through integrin proteins. Integrin availability on the plasma membrane, regulated by endosomal receptor uptake and recycling, has been extensively studied and regulates cell dynamics in various normal and pathological contexts1-5. In contrast, the role of integrin transport through the biosynthetic pathway has been considered primarily as a mechanism to replenish the receptor pool and too slow to influence cell dynamics6. Here, we adopted the RUSH (Retention Using Selective Hooks) assay to synchronize integrin anterograde transport from the endoplasmic reticulum (ER), allowing spatial and temporal analysis of newly synthesized receptor traffic. We observe that the delivery of new integrins to the plasma membrane is polarized in response to specific ECM ligands, facilitates integrin recruitment specifically to the membrane-proximal tip of focal adhesions (FA) and contributes to cell protrusion and FA growth. We explain the augmented adhesion growth using a computational molecular clutch model7, where increased integrin availability drives recruitment of additional integrins. Notably, a subset of newly synthesized integrins undergo rapid traffic from the ER to the cell surface to facilitate localized cell spreading, seemingly bypassing the Golgi. This unconventional secretion is dependent on cell adhesion and mediated by Golgi reassembling stacking proteins (GRASPs) association with the PDZ-binding motif in the integrin 5 cytoplasmic tail. This spatially targeted delivery of integrins through the biosynthetic pathway may propel cell dynamics by rapidly altering adhesion receptor availability, providing cells with an additional degree of plasticity to respond to their environment.

cell biology↗

bioRxiv2022

Proximity labelling identifies pro-migratory endocytic recycling cargo and machinery of the Rab4 and Rab11 families

Endocytic recycling controls the return of internalised cargos to the plasma membrane to coordinate their positioning, availability and downstream signalling. The Rab4 and Rab11 small GTPase families regulate distinct recycling routes, broadly classified as fast recycling from early endosomes (Rab4) and slow recycling from perinuclear recycling endosomes (Rab11), and both routes handle a broad range of overlapping cargos to regulate cell behaviour. We adopted a proximity labelling approach, BioID, to identify and compare the protein complexes recruited by Rab4a, Rab11a and Rab25 (a Rab11 family member implicated in cancer aggressiveness), revealing statistically robust protein-protein interaction networks of both new and well characterised cargos and trafficking machinery in migratory cancer cells. Gene ontological analysis of these interconnected networks revealed that these endocytic recycling pathways are intrinsically connected to cell motility and cell adhesion. Using a knock sideways relocalisation approach we were further able to confirm novel links between Rab11/25 and the ESCPE-1 and retromer multiprotein sorting complexes and identify new endocytic recycling machinery associated with Rab4, Rab11 and Rab25 that regulates cancer cell migration in 3D-matrix.

cell biology↗