Getting Mechanical Signals Across The Nucleus

Research in the Olivares laboratory focuses on understanding the mechanochemistry of protein complexes within the nuclear envelope important for converting forces felt by the cell into biochemically meaningful outputs such as the activation of gene transcription and chromosome positioning. Key questions driving research in the lab are:

1) How do LINC (linker of nucleoskeleton and cytoskeleton) complexes assemble?

LINC complexes comprise SUN (Sad1 and UNC-84) and KASH (Klarischt, ANC-1 and SYNE homology) domain-containing nesprin (nuclear envelope spectrin-repeat) proteins. SUN proteins bind to nucleoskeletal lamin proteins within the nucleus and span the inner nuclear membrane (INM) to interact with the KASH domain of nesprins in the perinuclear space (PNS or lumen). Nesprins span the outer nuclear membrane (ONM) and interact with the cytoskeleton. Several different LINC complexes form within the PNS consisting of SUN1-3 and nesprins 1-4 and appear to be dynamic in cells. We want to address the observed dynamic nature of these complexes by examining if inherent differences in thermodynamic and kinetic stabilities regulate complex assembly.

2) How does mechanical force affect LINC complex assembly and structure?

LINC complexes are proposed to withstand and transmit large mechanical loads over the nuclear envelope. However, nothing is known about which regions of LINC complex proteins are important for force transduction. For example, SUN proteins contain a long coiled-coil region important for trimerization in vitro and function in vivo (Sosa et al., Cell 2012). Sosa et al. propose that this region may serve to maintain the observed regular spacing between the ONM and INM that is lost upon SUN protein depletion (Crisp et al., J Cell Biol 2006). Does this triple helical arrangement impart unique mechanical properties to SUN proteins and LINC complexes in general? Under what loads do LINC complex proteins unfold and which parts unfold first? To address these questions, we are determining the forces at which mechanical unfolding occurs, the pathway of unfolding, the contribution of the coiled-coil region and oligomerization to unfolding, and how LINC complexes rupture using single-molecule force spectroscopy.