Biophysical Society Thematic Meeting

Mechanobiology of Disease

Poster Abstracts

47-POS

Board 47

LINC Complex Assembly Within The Nuclear Envelope Of Living Cells

Jared Hennen

, Cosmo A. Saunders

, Jochen D. Mueller

GW Gant Luxton

University of Minnesota, Minneapolis, MN, USA,

University of Minnesota, Minneapolis, MN,

USA.

LINC (linker of nucleoskeleton and cytoskeleton) complexes enable the direct transmission of

mechanical forces across the nuclear envelope and into the nucleoplasm, which is required for

wholesale positioning of meiotic chromosomes and nuclei. LINC complexes conserved

molecular bridges that span the nuclear envelope and consist of the inner and outer nuclear

membrane KASH and SUN proteins, respectively. KASH proteins interact with the cytoskeleton

within the cytoplasm, while in the nucleoplasm SUN proteins interact with nuclear lamins and

chromatin. Within the PNS (perinuclear space), KASH and SUN proteins directly interact to

form the core of the LINC complex. Recent structural studies revealed that SUN2 forms a

mushroom-like homo-trimeric structure with a stalk of three coiled-coils and a globular head that

recruits three KASH proteins that bind in three deep hydrophobic grooves formed between

adjacent SUN2 protomers. Despite this significant structural insight into LINC complex

assembly, the in vivo relevance and conservation of SUN homo-trimerization remain unclear.

Here, we apply z-scan FFS (fluorescence fluctuation spectroscopy), a biophysical imaging-based

approach with single-molecule sensitivity, to quantify interactions within the PNS of living cells

by brightness analysis. Consistent with in vitro studies, we demonstrate the existence of SUN2

homo-trimers within the PNS and identify structural requirements for SUN2 homo-trimerization.

We then show that within the PNS, SUN1 can form higher-order homo-oligomers than SUN2.

Finally, we uncover environment-dependent differences in SUN protein oligomerization

suggestive of previously unidentified mechanisms for the regulation of LINC complex assembly

and function. Taken together, our results establish z-scan FFS as a powerful tool for studying

LINC complex assembly within the nuclear envelope of living cells. This information will be

critical for understanding the functional consequences of mutations in KASH and SUN proteins

associated with human diseases including cancer and muscular dystrophies.