Active Microtubule-Actin Crosstalk Mediated by a Nesprin-2G-Kinesin Complex

Nesprins are integral membrane proteins that physically couple the nucleus and cytoskeleton. Nesprin-2 Giant (N2G) stands out for its extensive cytoplasmic domain, which contains tandem N-terminal actin-binding calponin-homology domains followed by >50 spectrin repeats and a C-terminal outer nuclear membrane-spanning KASH domain. N2Gs KASH domain interacts with the inner nuclear membrane, lamina-binding SUN proteins within the perinuclear space, forming a linker of nucleoskeleton and cytoskeleton (LINC) complex. Additionally, N2G contains a conserved W-acidic LEWD motif that enables the direct interaction with kinesin-1s light chain, indicating N2Gs involvement with both actin and microtubules. The absence of N2G leads to embryonic lethality in mice, while cellular assays highlight N2Gs role in nuclear positioning across diverse biological contexts. However, the precise mechanisms underlying N2G-mediated nucleocytoskeletal coupling remain unclear. Here we study N2Gs interactions with F-actin and kinesin-1, revealing its functions as an F-actin bundler, a kinesin-1-activating adapter, and a mediator of active cytoskeletal crosstalk. Along with MAP7 proteins, N2G directly links active kinesin-1 motors to F-actin, facilitating actin transport along microtubule tracks. These findings shed light on N2Gs dynamic role as a crosslinker between actin and microtubule cytoskeletons, offering insights into nuclear movement, a fundamental cellular process.