Tracking live-cell single-molecule dynamics enables measurements of heterochromatin-associated protein-protein interactions

Visualizing and measuring molecular-scale interactions in living cells represents a major challenge, but recent advances in single-molecule super-resolution microscopy are bringing us closer to achieving this goal. Single-molecule super-resolution microscopy enables high-resolution and sensitive imaging of the positions and movement of molecules in living cells. HP1 proteins are important regulators of gene expression because they selectively bind and recognize H3K9 methylated (H3K9me) histones to form heterochromatin-associated protein complexes that silence gene expression, but several important mechanistic details of this process remain unexplored. Here, we extended live-cell single-molecule tracking studies in fission yeast to determine how HP1 proteins interact with their binding partners in the nucleus. We measured how genetic perturbations that affect H3K9me alter the diffusive properties of HP1 proteins and their binding partners, and we inferred their most likely interaction sites. Our results demonstrate that H3K9 methylation spatially restricts HP1 proteins and their interactors, thereby promoting ternary complex formation on chromatin while simultaneously suppressing off-chromatin binding. As opposed to being an inert platform to direct HP1 binding, our studies propose a novel function for H3K9me in promoting ternary complex formation by enhancing the specificity and stimulating the assembly of HP1-protein complexes in living cells. Visualizing molecular-scale interactions in living cells is challenging, but advances in single-molecule super-resolution microscopy enable high-resolution imaging of molecular positions of proteins and their motions within cells. HP1 proteins bind to H3K9 methylated histones to form complexes that silence gene expression. Here, we tracked single HP1 proteins and their binding partners to measure when and where they form complexes in live fission yeast cells. Genetic perturbations enabled us to connect their motions to specific changes in their cellular properties. Surprisingly, we noted that HP1 proteins preferentially form ternary complexes with their binding partners at sites of H3K9me. This work proposes a novel function for chromatin and shows how H3K9 methylation spatially restricts HP1-associated complex formation while suppressing off-chromatin binding. Graphical Abstract