  Over recent years, super-resolution and single-molecule imaging methods have
delivered unprecedented details on the nanoscale organization and dynamics of
individual molecules in different contexts. Yet, visualizing single-molecule
processes in living cells with the required spatial and temporal resolution
remains highly challenging. Here, we report on an analytical approach that
extracts such information from live-cell single-molecule imaging at
high-labeling densities using standard fluorescence probes. Our
high-density-mapping (HiDenMap) methodology provides single-molecule nanometric
localization accuracy together with millisecond temporal resolution over
extended observation times, delivering multi-scale spatiotemporal data that
report on the interaction of individual molecules with their dynamic
environment. We validated HiDenMaps by simulations of Brownian trajectories in
the presence of patterns that restrict free diffusion with different
probabilities. We further generated and analyzed HiDenMaps from single-molecule
images of transmembrane proteins having different interaction strengths to
cortical actin, including the transmembrane receptor CD44. HiDenMaps uncovered
a highly heterogenous and multi-scale spatiotemporal organization for all the
proteins that interact with the actin cytoskeleton. Notably, CD44 alternated
between periods of random diffusion and transient trapping, likely resulting
from actin-dependent CD44 nanoclustering. Whereas receptor trapping was dynamic
and lasted for hundreds of milliseconds, actin remodeling occurred at the
timescale of tens of seconds, coordinating the assembly and disassembly of CD44
nanoclusters rich regions. Together, our data demonstrate the power of
HiDenMaps to explore how individual molecules interact with and are organized
by their environment in a dynamic fashion.
