Project Grant R35GM155264

QUANTITATIVE MOLECULAR IMAGING IN ENGINEERED CELLULAR MICROENVIRONMENTS TO STUDY AND CONTROL DIRECTIONAL MECHANOSENSING - PROJECT SUMMARY DIRECTIONAL SENSING OF FORCE PLAYS A CRITICAL ROLE IN A WIDE VARIETY OF PHYSIOLOGIC PROCESSES INCLUDING MIGRATION, ANGIOGENESIS, MORPHOGENESIS, AND MECHANICAL HOMEOSTASIS. AN IMPORTANT ASPECT OF THIS FORCE SENSING IS THAT MANY OF THESE FORCES ARE DYNAMIC IN NATURE, REQUIRING THAT CELLS CONSTANTLY PROBE, SENSE, AND RESPOND TO FORCE IN BOTH TIME AND SPACE USING FORCE SENSITIVE PROTEINS. WHILE THESE FORCES EXIST AT THE TISSUE SCALE, THEY ARE SENSED THROUGH MOLECULAR DEFORMATIONS AT THE PROTEIN SCALE THAT DRIVE CHANGES IN BINDING, REINFORCEMENT, AND DOWNSTREAM SIGNALING. DESPITE THE CRITICAL IMPORTANCE OF DIRECTIONAL FORCE SENSING, THE MOLECULAR MECHANISMS THAT ALLOW FOR THIS BEHAVIOR ARE UNCLEAR. WHILE MANY TOOLS NOW EXIST FOR MEASURING BIOPHYSICAL FORCES AND DIFFERENT LENGTH SCALES, THERE ARE SEVERAL UNRESOLVED QUESTIONS WITH RESPECT TO THE SCALE AT WHICH THIS DIRECTIONAL FORCE SENSING OCCURS. RECENT WORK HAS INDICATED THAT MECHANOSENSITIVE MOLECULES IN THE CYTOSKELETON DISPLAY DIRECTION DEPENDENT REINFORCEMENT OF BOND LIFETIMES AT THE MOLECULAR SCALE. WHILE THIS BEHAVIOR HAS THE POTENTIAL TO DRIVE DIRECTIONAL FORCE SENSING AT THE CELLULAR AND TISSUE SCALES, TOOLS FOR MEASURING THE ORIENTATION OF MOLECULAR FORCES IN LIVING CELLS ARE LIMITED. ONE ESTABLISHED METHOD FOR MEASURING ORIENTATION, POLARIZED FLUORESCENCE MICROSCOPY, IS LIMITED BY ITS LACK OF INTRACELLULAR FORCE AND DYNAMICS INFORMATION. THE GOAL OF THIS WORK IS TO DEVELOP METHODS FOR SIMULTANEOUS MEASUREMENT OF THESE DYNAMIC FORCES AND MATERIAL SYSTEMS FOR INTERROGATION OF CELLULAR RESPONSES TO DIRECTIONAL MECHANICAL CUES. TO ACCOMPLISH THIS, THIS RESEARCH PROGRAM WILL CONSIST OF THREE RESEARCH THEMES. THEME 1: DO MOLECULAR LEVEL FORCES AND FORCE ORIENTATIONS DRIVE EMERGENT DIRECTIONAL MECHANOSENSING BEHAVIORS AT THE CELLULAR SCALE? WE WILL DEVELOP IMAGING AND ANALYSIS METHODS FOR SIMULTANEOUS MEASUREMENT OF MULTIPLE BIOPHYSICAL VARIABLES IN LIVING CELLS THAT ALLOW FOR CROSS-CORRELATION OF MOLECULAR MEASUREMENTS WITH EMERGENT BEHAVIORS. THEME 2: CAN WE ENGINEER ENVIRONMENTS TO MANIPULATE AND STUDY DIRECTIONAL MECHANOSENSING BEHAVIORS AT THE MOLECULAR AND CELLULAR SCALE? BY ENGINEERING CELLULAR MICROENVIRONMENTS WITH INDEPENDENTLY CONTROLLABLE DIRECTIONAL CUES THAT DRIVE DUROTAXIS AND CONTACT GUIDANCE, WE WILL INVESTIGATE THE MOLECULAR BASIS OF THESE PHENOMENA. THEME 3: ARE ENVIRONMENTAL DEFORMATIONS DIRECTIONALLY SENSED THROUGH INDIVIDUAL PROTEINS WITH DIRECTIONALLY REINFORCED DOMAINS? BY DEVELOPING SYSTEMS FOR HIGH RESOLUTION ANALYSIS OF CELLULAR DEFORMATION MECHANOSENSING USING ENGINEERED LAMINATES WITH INDEPENDENTLY CONTROLLABLE STIFFNESS AND POISSON'S RATIO, WE WILL ASSESS BIOPHYSICAL CHANGES AT THE MOLECULAR AND CELLULAR SCALES IN RESPONSE TO DIRECTIONAL DEFORMATION FIELDS. THE COMBINED IMAGING MODALITIES AND MATERIAL SYSTEMS DEVELOPED IN THIS PROPOSAL WILL ALLOW FOR DETAILED DISSECTION OF DIRECTIONAL FORCE SENSING AT THE MOLECULAR LEVEL, PROVIDING AN IMPORTANT NEW TOOLSET FOR INTERROGATING AND MANIPULATING MOLECULAR MECHANISMS OF DIRECTIONAL FORCE SENSING.