The γ-tubulin meshwork assists in the recruitment of PCNA to chromatin in mammalian cells

Abstract

Changes in the location of γ-tubulin ensure cell survival and preserve genome integrity. We investigated whether the nuclear accumulation of γ-tubulin facilitates the transport of proliferating cell nuclear antigen (PCNA) between the cytosolic and the nuclear compartment in mammalian cells. We found that the γ-tubulin meshwork assists in the recruitment of PCNA to chromatin. Also, decreased levels of γ-tubulin reduce the nuclear pool of PCNA. In addition, the γ-tubulin C terminus encodes a PCNA-interacting peptide (PIP) motif, and a γ-tubulin–PIP-mutant affects the nuclear accumulation of PCNA. In a cell-free system, PCNA and γ-tubulin formed a complex. In tumors, there is a significant positive correlation between TUBG1 and PCNA expression. Thus, we report a novel mechanism that constitutes the basis for tumor growth by which the γ-tubulin meshwork maintains indefinite proliferation by acting as an opportune scaffold for the transport of PCNA from the cytosol to the chromatin.

γ-Tubulin in microtubule nucleation and beyond

“…It has been reported that γ-tubulin binds PCNA and aids in its recruitment to chromatin in mammalian cells. A positive correlation between γ-tubulin and PCNA expression was found in all examined tumor types (Corvaisier et al, 2021). Finally, γ-tubulin is capable to modulate the anaphase-promoting complex/cyclosome (APC/C), which is a large protein complex with multiple subunits that is important for cell cycle regulation.…”

“…As described in the following section, γ-tubulin can also be found in cell nuclei. It has been proposed that γ-tubulin filaments may also play a structural role in nuclei (Corvaisier et al, 2021). Further studies are however needed to verify the presence of fibrillar γ-tubulin assemblies in different model systems, determine their composition, structure and decipher their cellular function(s).…”

Optimization of production of recombinant gamma-tubulin in bacteria

“…Materials pGEX2T- TUBG1 (Addgene Plasmid # 171967) [6] pET21- TUBG1 (Addgene Plasmid # 101825) [13] E. coli DH5α (gives high protein yield from pGEX2T- TUBG1 plasmid; Thermo Fisher Scientific, cat. no.…”

“…Acentrosomal and centrosome-associated microtubules nucleate from the minus end on a complex formed of TUBG and various TUBG complex proteins, known as the TUBG ring complex [3] . TUBG is a ubiquitously expressed protein that forms a novel cytoskeleton/nucleoskeleton named the γ-tubulin meshwork [ [4] , [5] – 6 ]. The meshwork can interact with various proteins, such as E2F1 [1] , lamin B1 [7] , Rad51 [8] , GCPs [3] , PCNA [6] , C53 [9] , and α- and β-tubulin dimers [ 2 , 3 ], and influences various cellular processes [ 4 , 10 , 11 ].…”

“…TUBG is a ubiquitously expressed protein that forms a novel cytoskeleton/nucleoskeleton named the γ-tubulin meshwork [ [4] , [5] – 6 ]. The meshwork can interact with various proteins, such as E2F1 [1] , lamin B1 [7] , Rad51 [8] , GCPs [3] , PCNA [6] , C53 [9] , and α- and β-tubulin dimers [ 2 , 3 ], and influences various cellular processes [ 4 , 10 , 11 ]. The cellular folding of TUBG depends on chaperonin TCP1, which has hindered the purification of denatured protein in bacteria, and consequently, the use of recombinant TUBG in analysis in vitro [12] .…”