PROJECT SUMMARY
Cells perform diverse processes, such as cell division, growth, motility, formation of adhesions, and tissue
morphogenesis, under a wide range of mechanical environments. Central to these processes are mechanical
forces, which may come from outside the cell or be generated internally and which are integrated with signaling
pathways to guide the cellular process. The cell's macromolecular cytoskeletal machinery, including the actin-
based myosin II motors and actin crosslinking proteins, assemble, function and then disassemble in response to
these forces and signaling pathways. This dynamic force-responsive assembly provides self-tuning of the
machinery, leading to natural positive and negative feedback and further allows mechanical inputs to be
converted into signaling outputs. Using Dictyostelium cells, we discovered that many of these components are
pre-assembled in the cytoplasm in the form of mechanoresponsive Contractility Kits (CKs), which allow for highly
efficient responses to force inputs. The CKs include myosin II, cortexillin I, IQGAP1, IQGAP2, plus several other
proteins that we know of. For this application, substantial published and unpublished data motivate the questions
to be answered, and our work extends from Dictyostelium to human proteins and model systems. We begin by
leveraging our suite of experimental and modeling platforms, including a new modeling framework called
SpringSaLaD, which allows for molecularly motivated, particle-based, stochastic simulations of biochemical
processes. Using SpringSaLaD, we are modeling the formation of CKs by drawing upon measured in vivo
concentrations, diffusion constants, and in vivo “apparent” KDs. From this model, we have made an initial list of
predictions about the features of the CKs, which we will test in Dictyostelium. We will also explore the kinetics of
assembly and disassembly of the CKs with and without mechanical force. For assembly, we will determine the
molecular basis for force-dependent assembly of the CKs and nonmuscle myosin II bipolar thick filament (BTF),
using interference scattering mass spectrometry. For disassembly, we will use magnetic tweezers to measure
the compliance within the BTF and then determine how this compliance restricts the activity of the myosin heavy
chain kinase (MHCKC for Dictyostelium and PKCzeta for NMIIB). We have also found that the setpoint of
mechanosensitive accumulation (mechanoaccumulation) by Dictyostelium myosin II and human NMIIB has an
optimum of 20% assembly fraction. Further, NMIIB's setpoint is cell type- and cell cycle stage-specific. We will
use the framework we have established to determine the consequences of setpoint positioning on cell behavior,
including NMIIB dynamics, cell division, and gene expression. We will incorporate this information into our
computational models for myosin II mechanoaccumulation, expanding the models to include the components of
the CKs. In sum, this research effort, which spans molecular to cellular scales combined with physical theory
development, will decipher key principles and mechanisms of force-dependent cytoskeletal assembly and the
impact on cell behavior.