Regulation of microtubule minus-end dynamics by CAMSAPs and Patronin

Abstract

The microtubule (MT) cytoskeleton plays an essential role in mitosis, intracellular transport, cell shape, and cell migration. The assembly and disassembly of MTs, which can occur through the addition or loss of subunits at the plus-or minus-ends of the polymer, is essential for MTs to carry out their biological functions. A variety of proteins act on MT ends to regulate their dynamics, including a recently described family of MT minus-end binding proteins called calmodulin-regulated spectrin-associated protein (CAMSAP)/Patronin/Nezha. Patronin, the single member of this family in Drosophila, was previously shown to stabilize MT minusends against depolymerization in vitro and in vivo. Here, we show that all three mammalian CAMSAP family members also bind specifically to MT minus-ends and protect them against kinesin-13-induced depolymerization. However, these proteins differ in their abilities to suppress tubulin addition at minus-ends and to dissociate from MTs. CAMSAP1 does not interfere with polymerization and tracks along growing minus-ends. CAMSAP2 and CAMSAP3 decrease the rate of tubulin incorporation and remain bound, thereby creating stretches of decorated MT minus-ends. By using truncation analysis, we find that somewhat different minimal domains of CAMSAP and Patronin are involved in minus-end localization. However, we find that, in both cases, a highly conserved C-terminal domain and a more variable central domain cooperate to suppress minus-end dynamics in vitro and that both regions are required to stabilize minus-ends in Drosophila S2 cells. These results show that members of the CAMSAP/Patronin family all localize to and protect minus-ends but have evolved distinct effects on MT dynamics.tubulin polymerization | cytoskeletal regulation | TIRF microscopy M icrotubules (MTs) are cellular polymers that are important for a variety of functions, including cargo transport and mitotic spindle formation. MTs are composed of dimers of α-and β-tubulin that assemble head-to-tail, creating a polar protofilament. Protofilaments then assemble laterally to form the canonical 13-protofilament MT structure, with β-tubulin exposed at fast-growing plus-ends and α-tubulin exposed at slow-growing minus-ends (1). MTs exhibit an intriguing property termed "dynamic instability," whereby the polymer can abruptly switch between episodes of net growth and shrinkage (2). The rates of growth and shrinkage as well as the frequency of transitions between these two states are regulated by numerous MT-associated proteins, many of which bind to the ends of the polymer (3, 4).The dynamics of MT plus-ends are regulated by a well-characterized network of plus-end tracking proteins (+TIPs) (5). End-binding proteins recognize a tubulin conformation unique to the growing ends of MTs and can affect the dynamics of plusends by intrinsically altering the structure of the MT end (6-8) as well as recruiting other interacting proteins (9). In contrast, TOG domain-containing proteins, such as XMAP215, promote MT growth and have been suggested ...

CAMSAP3 orients the apical-to-basal polarity of microtubule arrays in epithelial cells

“…S4 for further characterization of the CKK domain-deleted CAMSAP3.) Given the previous observation that CAMSAP3 selectively binds to the minus ends of microtubules (11)(12)(13)(14)(15)(16), our results suggest that CAMSAP3, concentrated at apical cortices, tethers microtubules to these sites through the binding to their minus-ends, ensuring the longitudinal alignment of this cytoskeleton, and that this function of CAMSAP3 is abolished in the absence of the CKK domain.…”

“…S6). Given the previous finding that CAMSAP3 or its Drosophila homolog Patronin (11,(13)(14)(15)19) binds the minus ends of microtubules, it is likely that CAMSAP3 holds microtubules via this mechanism at the apical cortex. Arrays of microtubules still formed in the absence of functional CAMSAP3, however.…”

“…The resultant mutant mice expressed a C terminustruncated CAMSAP3 (Fig. S1 B-E), which lacks the CKK domain essential for the binding of this molecule to the microtubule (11,12,14,15). RT-PCR analysis confirmed that mRNA transcribed from the mutated gene does not cover the exons that encode the CKK domain ( Fig.…”

“…CAMSAP3 (also known as Nezha) is a member of the calmodulin-regulatedspectrin-associated proteins (CAMSAP)/Nezha/Patronin family proteins, which bind and stabilize the minus-ends of microtubules (11)(12)(13)(14)(15)(16)(17)(18). In cultured mammalian cells, CAMSAP proteins have been shown to stabilize noncentrosomal microtubules in the cytoplasm or cell junctions (11,14,19,20), suggesting their possible involvement in the spatial regulation of microtubule assembly in polarized cells, such as epithelial-specific longitudinal microtubule alignment.…”

NuMA Recruits Dynein Activity to Microtubule Minus-Ends at Mitosis

“…NuMA can target dynein-dynactin to the cell cortex (Lechler and Fuchs, 2005;Nguyen-Ngoc et al, 2007) and thus could be one such adaptor. However, in vitro NuMA has shown no direct affinity for minus-ends specifically, binding all along the microtubule lattice (Du et al, 2002;Forth et al, 2014;Haren and Merdes, 2002) or at both ends (Seldin et al, 2016), unlike three proteins known to interact directly with minus-ends at mitosis: gTuRC (Zheng et al, 1995), CAMSAP1 (Akhmanova and Hoogenraad, 2015;Hendershott and Vale, 2014;Jiang et al, 2014) and KANSL1/3 (Meunier et al, 2015). In cells, NuMA is thought to require dynein activity to carry it to minus-ends and spindle poles (Merdes et al, 2000), where it anchors spindle microtubules (Dionne et al, 1999;Gaglio et al, 1995;Heald et al, 1997;Silk et al, 2009).…”