Cytoskeleton, Nucleus and Integrin Recycling in Cell Migration

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SUMMARY This application for an Administrative Supplement requests an epifluorescence, FRET and TIRF microscope to pursue the goals of the parent R35 grant to understand two aspects of cell migration for which we have limited understanding: nuclear positioning and integrin recycling. Both these processes are essential for cell migration, yet how they contribute at a mechanistic level to cell migration is lacking. The parent R35 grant seeks to build on results previously obtained in two previous projects in the Gundersen laboratory supported by NIGMS. For nuclear positioning, we will examine how the linker of nucleoskeleton and cytoskeleton (LINC) complex is mechanically reinforced to resist the large forces necessary to move the nucleus and will use and develop new FRET tension sensors to directly measure forces on the nucleus. We will determine how the LINC complex selects actin filament or microtubules for nuclear movement and the functional consequences of these interactions for different modes of cell migration. We will test new hypotheses that the nucleus functions as a “tension resistor” for actin filaments and as a polarity factor for microtubule trafficking. For integrin recycling, we will test the overall hypothesis that recycled integrins travel in an active conformation and that this seeds new adhesion formation in a polarized manner near the leading edge. We will use new integrin probes and a new integrin recycling system coupled with TIRF microscopy to identify the intracellular pathway of recycling integrins, their sites of integrin exocytosis relative to newly formed adhesions. We will also use TIRF microscopy to determine whether recycled integrin derives from the cell rear during migration and contributes to nascent adhesion formation near the leading edge. We will identify the microtubule machinery that we hypothesize is responsible for the polarized reformation of adhesions from recycled integrins and test the possibility that the recycled integrin plays a role in integrin signaling. We will explore whether these two aspects of cell migration can be linked by determining with FRET imaging of tension sensors whether the nucleus mechanically engages substrate adhesions. Our proposed studies on these two processes will advance understanding of the basic mechanisms of cell migration and potential identify new targets for intervening in cases when cell migration goes awry. The proposed studies will also provide fertile ground for postdoctoral fellows and graduate students to advance their training and develop their own projects.