A structural model for microtubule minus-end recognition and protection by CAMSAP proteins

Abstract

CAMSAP and Patronin family members regulate microtubule minus-end stability and localization and thus organize non-centrosomal microtubule networks, which are essential for cell division, polarization and differentiation. Here, we show that the C-terminal CKK domain of CAMSAPs is widely present among eukaryotes and autonomously recognizes microtubule minus ends. Through a combination of structural approaches, we uncover how mammalian CKK binds between two tubulin dimers at the inter-protofilament interface on the outer microtubule surface. In vitro reconstitution assays combined with high resolution fluorescence microscopy and cryo-electron tomography suggest that CKK preferentially associates with the transition zone between curved protofilaments and the regular microtubule lattice. We propose that minus-end-specific features of the inter-protofilament interface at this site form the basis for CKK’s minus-end preference. The steric clash between microtubule-bound CKK and kinesin motors explains how CKK protects microtubule minus ends against kinesin-13-induced depolymerization and thus controls the stability of free microtubule minus ends.

Structural determinants of microtubule minus end preference in CAMSAP CKK domains

“…Can CKK binding to different MT protofilament architectures shed further light on the mechanism of minus end recognition? Intriguingly, we also previously identified CKKs in the amoeboflagellate N. gruberi and the potato blight fungus P. infestans that had lost the binding preference for MT minus ends that was proposed to be present in CKK domains from the last eukaryotic common ancestor 2 . Can a comparison of CKK domains with and without minus-end binding specificity also provide insight into MT minus-end recognition?…”

“…Strep-GFP-tagged human CAMSAP1 CKK (residues 1474-C) and Naegleria gruberi CKK (residues 612-C, reference sequence XM_002675733.1) were prepared as described previously (Atherton et al, 2017). Briefly, both proteins were expressed in HEK293T cells using a modified pTT5 expression vector (Addgene no.…”

“…The ends of MTs are important sites of conformational diversity and are often points of communication between the MT cytoskeleton and other cellular components, such as membranes, organelles, centrosomes and chromosomes 1 . The exact conformation(s) of MT ends is an ongoing source of debate, but current evidence points to their being composed of zones with distinct and dynamic tubulin conformations [2][3][4][5][6][7] . MT minus ends were long thought to be static and structurally homogeneous, capped by g-TuRCs and buried at MT organizing centres.…”

A perinuclear microtubule-organizing center controls nuclear positioning and basement membrane secretion

“…How Patronin functions to assemble MTs at the ncMTOC remains incompletely understood. Patronin/CAMSAP stabilizes MT minus-ends and antagonizes the activity of Kinesin-13 family depolymerases (Goodwin and Vale, 2010;Hendershott and Vale, 2014;Atherton et al, 2017). However, this antagonism does not prevail at the fat body ncMTOC because Kinesin-13 (Klp10A) knockdown, or other MT depolymerases Klp59C or Klp59D did not suppress the reduced circumferential MTs in Patronin knockdown cells ( Figure 17E, F).…”

“…The XMAP215/Msps/Stu2/ Dis1/Alp14/ZYG-9/ch-TOG/MOR1 is an ancient family of MT polymerases that was recently identified as a new in vivo MT nucleator at centrosomes and spindle pole bodies through its association with g-tubulin (Flor-Parra et al, 2018;Gunzelmann et al, 2018;Thawani et al, 2018); a role for the MT polymerase at nucleating MTs at ncMTOCs has not been reported. Additionally, the recently identified CAMSAP/Patronin family of MT minus-end proteins protects MT minusends from Kinesin-13 family depolymerases (Goodwin and Vale, 2010;Hendershott and Vale, 2014;Atherton et al, 2017). CAMSAP/Patronin has emerged as a critical player at ncMTOCs via unclear mechanisms.…”