Biology subjects

Hammer, D. A.

Publications and source records attributed to Hammer, D. A..

4 recordsLinked to original sources

bioRxiv2025

Characterizing Traction Forces in Upstream-Migrating Hematopoietic-like KG1a Cells Under Shear Flow

Cell migration is critical to leukocyte function, enabling leukocytes to patrol tissues and respond to inflammatory cues. Upstream migration is a distinct form of cell motility which enables leukocytes to move against the direction of fluid flow on Intercellular Adhesion Molecule-1 (ICAM-1) surfaces. Upstream migration is mediated by the leukocyte integrin, Lymphocyte Function-Associated Antigen-1 (LFA-1). While this behavior has been observed across multiple immune cell types, the mechanical forces underlying upstream migration have not been measured. Here, we demonstrate the use of Traction Force Microscopy (TFM) to quantify spatiotemporal patterns of force generation during upstream migration of KG1a cells, a hematopoietic progenitor cell line that exhibits robust upstream migration on ICAM-1 functionalized hydrogels. Under static (no-flow) conditions, KG1a cells displayed random motility and traction profiles that varied with time. In contrast, cells exposed to shear flow generated persistent, polarized tractions aligned with the axis of migration. Population analysis showed that maximum RMS traction forces were significantly elevated during upstream migration compared to static conditions (mean: 428.5 {+/-} 63.0 nN vs 220.8 {+/-} 22.2 nN, p = 0.0078), as were average RMS forces (mean: 82.6 {+/-} 12.9 nN vs 45.9 {+/-} 4.4 nN, p = 0.0184), while minimum force values remained comparable between groups. These findings indicate a specific amplification of stresses required to overcome applied forces during upstream migration. By integrating single-cell and population-level force analyses, this study defines upstream migration as a mechanically reinforced state characterized by amplified, directionally coherent traction dynamics. Our methods enable future dissection of the molecular regulators that coordinate force generation with migration under flow. SIGNIFICANCEUpstream migration is a critical yet poorly understood mode of immune cell motility. Here, we use traction force microscopy under physiologically relevant shear stresses to provide the first quantitative characterization of traction dynamics during upstream migration. We show that upstream migrating cells generate significantly greater average and peak forces than cells under static conditions, revealing a distinct mechanical program for directed migration against flow. These findings establish a platform for dissecting the molecular regulators of force generation in immune cells and set the stage for future perturbation-based studies aimed at understanding how mechanical forces shape immune cell trafficking and vascular navigation.

biophysics↗

bioRxiv2024

Assembly of Hierarchical Multiphase Condensates Using Designer Surfactant Proteins

Biomolecular condensates are complex fluids formed by liquid-liquid phase separation of macromolecules. Similar to other types of soft matter, they feature a range of biophysical properties that distinguish them from the cellular milieu. As a separate phase, they have an identifiable interface that dictates their interaction with the cytoplasm and other membraneless organelles. In this work, we engineer the interface of condensates to build novel hierarchical mesoscale structures from two immiscible disordered proteins: the RGG domain of LAF-1, a RNA-processing protein involved in germ granule assembly and the low complexity domain (LC) of FUS, an RNA binding protein whose aggregation is implicated in age-related neurodegeneration. RGG and FUS LC do not co-partition with one another and instead form discrete protein-rich condensed phases. Despite their apparent immiscibility, we identified conditions that can promote hierarchical assembly, either kinetically trapping one phase in the other, or using a designer surfactant protein that reduces the interfacial tension between the two phases. In addition, we studied factors that impact condensate miscibility and structure formation, including surface properties and viscoelasticity. This study probes the principles that underlie formation and assembly of complex structures from biomolecular condensates and provides a strategy for designing synthetic multiphasic materials capable of spatial partitioning.

biophysics↗

bioRxiv2020

LFA-1 signals to promote actin polymerization and upstream migration in T cells

T cell entry into inflamed tissue requires firm adhesion, cell spreading, and migration along and through the endothelial wall. These events require the T cell integrins LFA-1 and VLA-4 and their endothelial ligands ICAM-1 and VCAM-1, respectively. T cells migrate against the direction of shear flow on ICAM-1 and with the direction of shear flow on VCAM-1, suggesting that these two ligands trigger distinct cellular responses. However, the contribution of specific signaling events downstream of LFA-1 and VLA-4 has not been explored. Using primary mouse T cells, we found that engagement of LFA-1, but not VLA-4, induces cell shape changes associated with rapid 2D migration. Moreover, LFA-1 ligation results in activation of the PI3K and ERK pathways, and phosphorylation of multiple kinases and adaptor proteins, while VLA-4 ligation triggers only a subset of these signaling events. Importantly, T cells lacking Crk adaptor proteins, key LFA-1 signaling intermediates, or the ubiquitin ligase cCbl, failed to migrate against the direction of shear flow on ICAM-1. These studies identify novel signaling differences downstream of LFA-1 and VLA-4 that drive T cell migratory behavior. Summary StatementInflammatory responses require leukocyte migration along the vascular wall. We show that signaling from {beta}2, but not {beta}1, integrins induces cytoskeletal changes needed for upstream migration under shear flow.

cell biology↗

bioRxiv2020

Identifying Sequence Perturbations to an Intrinsically Disordered Protein that Determine Its Phase Separation Behavior

Phase separation of intrinsically disordered proteins (IDPs) commonly underlies the formation of membraneless organelles, which compartmentalize molecules intracellularly in the absence of a lipid membrane. Identifying the protein sequence features responsible for IDP phase separation is critical for understanding physiological roles and pathological consequences of biomolecular condensation, as well as for harnessing phase separation for applications in bio-inspired materials design. To expand our knowledge of sequence determinants of IDP phase separation, we characterized variants of the intrinsically disordered RGG domain from LAF-1, a model protein involved in phase separation and a key component of P granules. Based on a predictive coarse-grained IDP model, we identified a region of the RGG domain that has high contact probability and is highly conserved between species; deletion of this region significantly disrupts phase separation in vitro and in vivo. We determined the effects of charge patterning on phase behavior through sequence shuffling. By altering the wild-type sequence, which contains well-mixed charged residues, to increase charge segregation, we designed sequences with significantly increased phase separation propensity. This result indicates the natural sequence is under negative selection to moderate this mode of interaction. We measured the contributions of tyrosine and arginine residues to phase separation experimentally through mutagenesis studies and computationally through direct interrogation of different modes of interaction using all-atom simulations. Finally, we show that in spite of these sequence perturbations, the RGG-derived condensates remain liquid-like. Together, these studies advance a predictive framework and identify key biophysical principles of sequence features important to phase separation. Significance StatementMembraneless organelles are assemblies of highly concentrated biomolecules that form through a liquid-liquid phase separation process. These assemblies are often enriched in intrinsically disordered proteins, which play an important role in driving phase separation. Understanding the sequence-to-phase behavior relationship of these disordered proteins is important for understanding the biochemistry of membraneless organelles, as well as for designing synthetic organelles and biomaterials. In this work, we explore a model protein, the disordered N-terminal domain of LAF-1, and highlight how three key features of the sequence control the proteins propensity to phase separate. Combining predictive simulations with experiments, we find that phase behavior of this model IDP is dictated by the presence of a short conserved domain, charge patterning, and arginine-tyrosine interactions.

biophysics↗