Membrane Curvature During Cell Migration

Abstract

Biological membranes serve as a physical barrier of cell and organelles. In addition, as biological membranes house numerous proteins, they also act as signaling hubs involved in the regulation of a wide array of cellular functions, including proliferation, differentiation, communication, and migration. Migrating cells can deform their plasma membrane to adapt to structural features in their environment, and reorganize their behaviors accordingly. In addition, when responding to extracellular chemical stimuli, cells are undergoing extensive endocytosis and exocytosis thereby continuously generating intracellular membrane curvatures However, the mechanisms by which cells sense and transmit outside structural features to generate specific responses remain mostly unknown. The work described in this thesis aims to assess the role of extracellular and intracellular membrane curvatures during two processes that have key relevance to human health: breast cancer invasion and neutrophil-mediated inflammation. Numerous studies suggest that structural features within the cancer cell microenvironment are key regulators of cancer invasion and metastasis. Using aligned nanoscale ridges and asymmetric sawtooth structures, I explored the effects of surface topography on the migration phenotype of multiple breast cancer cell lines. I discovered that asymmetric sawtooth structures unidirectionally bias the movement of breast cancer cells in a cell-type dependent manner. I went on to show that the biased migration is driven by unidirectional actin polymerization, and regulated by distinct cortical plasticity and focal adhesion patterns. Together, this work highlights the significance of extracellular matrix (ECM) topographies in cancer invasion and suggests that cell-ECM interactions are potential target to prevent cancer dissemination. In the context of tissue injury or inflammation, nearby neutrophils migrate directionally to inflamed/injured sites and trigger a dramatic swarm-like recruitment of distant neutrophils by secreting the secondary chemoattractant leukotriene B4 (LTB4) - a process referred to as signal relay. In this context, I studied how extracellular topographies regulate neutrophil recruitment. I found that neutrophils plated on nanoridges spread and exhibit rapid calcium flashes in the absence of chemical stimuli and adhesion ligands. Remarkably, I further showed that these responses are regulated by cell membrane curvatures, as I found that neutrophil activation is mediated by Beta2 integrins, and that active Beta2 integrins cluster on the side walls of the nanoridges. In a parallel project, I studied how neutrophils package and release LTB4. LTB4 is synthesized from arachidonic acid (AA) through the sequential action of the 5-lipoxygenase (5-LO) and its associated activating protein (FLAP). LTB4 and its synthesizing enzymes are packaged and released in extracellular vesicles called exosomes. The biogenesis of these vesicles is initiated at the nuclear envelope through the hydrolysis of nuclear AA, and the genesis of ceramide microdomains via the activation of the neutral sphingomyelinase. I aimed to visualize the structural organization of FLAP in defined lipid environments and assess if they form membrane curvatures similar to what is observed at the nuclear envelope of activated neutrophils. By reconstituting FLAP into lipids and visualizing the membrane organization of the protein:lipid assemblies using negative staining electron microscopy (EM) and cryogenic EM, I found that FLAP is assembled into specific “wagon-wheel” structures with potentially two membrane layers and multiple protrusions between the two layers. Collectively, the findings presented in my thesis reveal a novel role for membrane curvatures in regulating various physiological and pathological processes, and provide guidance for future research.