Molecular basis for CPAP-tubulin interaction in controlling centriolar and ciliary length

Abstract

Centrioles and cilia are microtubule-based structures, whose precise formation requires controlled cytoplasmic tubulin incorporation. How cytoplasmic tubulin is recognized for centriolar/ciliary-microtubule construction remains poorly understood. Centrosomal-P4.1-associated-protein (CPAP) binds tubulin via its PN2-3 domain. Here, we show that a C-terminal loop-helix in PN2-3 targets β-tubulin at the microtubule outer surface, while an N-terminal helical motif caps microtubule's α-β surface of β-tubulin. Through this, PN2-3 forms a high-affinity complex with GTP-tubulin, crucial for defining numbers and lengths of centriolar/ciliary-microtubules. Surprisingly, two distinct mutations in PN2-3 exhibit opposite effects on centriolar/ciliary-microtubule lengths. CPAPF375A, with strongly reduced tubulin interaction, causes shorter centrioles and cilia exhibiting doublet- instead of triplet-microtubules. CPAPEE343RR that unmasks the β-tubulin polymerization surface displays slightly reduced tubulin-binding affinity inducing over-elongation of newly forming centriolar/ciliary-microtubules by enhanced dynamic release of its bound tubulin. Thus CPAP regulates delivery of its bound-tubulin to define the size of microtubule-based cellular structures using a ‘clutch-like' mechanism.

Once and only once: mechanisms of centriole duplication and their deregulation in disease

“…Consistent with structural studies that showed CPAP controls the speed of microtubule growth during centriole assembly 37–39 , overexpression of CPAP or its interaction partners, CEP120 and SPICE1 ( Spi ndle and Ce ntriole Associated Protein 1), triggers the assembly of excessively long centrioles 45,51–54 . Centriole length can also be modulated by deregulation of proteins implicated in building the distal halves of centrioles, including the WD40 protein POC1 ( P roteome O f C entriole protein 1) 55 , the centrin-binding protein hPOC5 56 , or the microtubule binding protein CEP295 (Ana1 in Drosophila ) 57,58 .…”

“…The exact role of CEP135 in cartwheel formation in vertebrates therefore remains unclear and no homolog of Bld10p has been identified in Caenorhabditis elegans . Finally, the deposition of microtubules onto the cartwheel clearly requires CPAP ( C entrosomal P 4.1- A ssociated P rotein)/hSAS-4) 3738,39 .…”

“…Further downstream events in centriole biogenesis remain to be fully elucidated, but there is evidence that CEP135 serves to connect SAS-6 to CPAP and outer microtubules of the microtubule triplets 34 . During centriole elongation CPAP then regulates the growth of centriolar microtubules 37–39,52 , which are inserted underneath a cap of CP110 36 . Interestingly, CPAP also interacts with STIL and it will be important to understand how CPAP and STIL modulate each other’s activities 32,111–113 .…”

A transient microtubule‐based structure uncovers a new intrinsic asymmetry between the mother centrioles in the early Drosophila spermatocytes

“…Surprisingly, in addition to identifying two pairs of small dots for each germ cell, the anti Sas‐4 antibody also recognized distinct filaments (Figure a) that overlapped the Tbs (Figure a, inset). To exclude that this signal was due to nonspecific cross‐reactivity of the Sas‐4 monoclonal antibody we also examined young primary spermatocytes obtained from a Drosophila strain expressing a GFP tagged Sas‐4 (Zheng et al, ). Endogenous Sas‐4 was found on both centrioles and Tbs (Figure b inset).…”

“…Rootletin‐GFP (Chen et al, ); Unc‐GFP (Baker et al, ); Ana1‐GFP (Blachon et al, ); Asl‐YFP (Varmark et al, ); Sas‐4‐GFP 4 (Zheng et al, ); Sas‐4 (Basto et al, ) and Klp10A (Peter et al, ) mutant lines. The Cnb loss of function was obtained as described in Gottardo et al ().…”