Cell movement through three-dimensional (3D) extracellular matrix (ECM) is an essential component of normal physiology and disease, including wound healing and tumor metastasis. Understanding how cells move through structurally diverse 3D matrices will be essential to design therapies aimed at controlling cell migration in the body. During 3D migration, both metastatic tumor cells and wound healing fibroblasts are faced with the same problem: how to efficiently move the bulky, stiff nucleus. While the power of actomyosin contractility is essential for cells to move their nuclei in 3D matrices, it is not understood how it is regulated by the ECM structure or coupled to the influx of water into the cells that is necessary to sustain high-pressure migration. An additional layer of complexity comes from the fact that the 3D matrix structure can govern actomyosin contractility to dictate the type of protrusions cells use to move (i.e. migratory plasticity). By understanding how the structure of the 3D ECM affects actomyosin contractility and water influx, we aim to create a conceptual framework to explain how and why human cells switch between distinct 3D migration mechanisms. We recently discovered that human cells moving in a linearly elastic 3D matrix rely on integrin-based cell-matrix adhesions and the power of actomyosin contractility to pull the nucleus forward, like a piston, and switch from using low-pressure lamellipodia to high-pressure lobopodial protrusions. This project will explore how the cell reprograms its intracellular architecture and polarity to power the nucleus, and thereby the cell through 3D matrices. To achieve these goals, we will focus on three key gaps in knowledge. Question 1: how is the nuclear piston pulling mechanism activated in response to the physical environment? While the nucleus can act as a mechanosensor to control contractility at the rear of the cell, the mechanosensor that regulates the pulling mechanism has not been identified. Question 2: How are organelles and endomembranes organized and transported past the nucleus in compartmentalized, pressurized cells using the piston mechanism? This question will help us understand how the secretion pathways identified addressing question 1 function in high- pressure, compartmentalized cells. Question 3: Once activated, how is the anterior contractility responsible for pulling the nucleus forward sustained over the hours to days that primary human fibroblasts sustain this mode of 3D migration? While contractility at the trailing edge is well known to push the nucleus forward, very little is understood about the longer-term regulation of the pulling forces generated by the piston mechanism. By addressing these questions, our research will establish how distinct pools to actomyosin contractility are linked to the control of cytoplasmic hydraulic pressure to achieve 3D motility. This enhanced understanding of the fundamental principles of directional 3D cell motility and migratory plasticity will lead to new therapeutic strategies to control normal and abnormal cell movement in the body.

Project Narrative The movement of single cells through three-dimensional tissue environments is essential for physiological processes like wound healing, but is a lethal characteristic of metastatic tumor cells. The goal of this research project is to understand how cell behavior and architecture is reprogrammed in response to the structure of the material they are moving through. This information will help develop new therapeutic strategies to control the movement of normal and abnormal cells in the body.