KASH proteins in the outer nuclear membrane comprise the cytoplasmic half of LINC complexes that connect nuclei to the cytoskeleton. Caenorhabditis elegans ANC-1, an ortholog of Nesprin-1/2, contains actin-binding and KASH domains at opposite ends of a long spectrin-like region. Deletion of either the KASH or calponin homology (CH) domains does not completely disrupt nuclear positioning, suggesting neither KASH nor CH domains are essential. Deletions in the spectrin-like region of ANC-1 led to significant defects, but only recapitulated the null phenotype in combination with mutations in the trans-membrane span. In anc-1 mutants, the ER, mitochondria, and lipid droplets were unanchored, moving throughout the cytoplasm. The data presented here support a cytoplasmic integrity model where ANC-1 localizes to the ER membrane and extends into the cytoplasm to position nuclei, ER, mitochondria, and likely other organelles in place.

Numerous proteins target lipid droplets (LDs) through amphipathic helices (AHs). It is generally assumed that AHs insert bulky hydrophobic residues in packing defects at the LD surface. However, this model does not explain the targeting of perilipins, the most abundant and specific amphipathic proteins of LDs, which are weakly hydrophobic. A striking example is Plin4, whose gigantic and repetitive AH lacks bulky hydrophobic residues. Using a range of complementary approaches, we show that Plin4 forms a remarkably immobile and stable protein layer at the surface of cellular or in vitro generated oil droplets, and decreases LD size. Plin4 AH stability on LDs is exquisitely sensitive to the nature and distribution of its polar residues. These results suggest that Plin4 forms stable arrangements of adjacent AHs via polar/electrostatic interactions, reminiscent of the organization of apolipoproteins in lipoprotein particles, thus pointing to a general mechanism of AH stabilization via lateral interactions.