Abstract
The centrosomal protein γ-tubulin complex protein 3 (Tubgcp3/GCP3) is required for the assembly of γ-tubulin small complexes (γ-TuSCs) and γ-tubulin ring complexes (γ-TuRCs), which play critical roles in mitotic spindle formation during mitosis. However, its function in vertebrate embryonic development is unknown. Here, we generated the zebrafish tubgcp3 mutants using the CRISPR/Cas9 system and found that the tubgcp3 mutants exhibited the small eye phenotype. Tubgcp3 is required for the cell cycle progression of retinal progenitor cells (RPCs), and its depletion caused cell cycle arrest in the mitotic (M) phase. The M-phase arrested RPCs exhibited aberrant monopolar spindles and abnormal distributed centrioles and γ-tubulin. Moreover, these RPCs underwent apoptosis finally. Our study provides the in vivo model for the functional study of Tubgcp3 and sheds light on the roles of centrosomal γ-tubulin complexes in vertebrate development.
Highlights
Centrosome, the major microtubule-organizing center (MTOC) in vertebrate cells, provides a major site for microtubule (MT) nucleation and plays key roles in bipolar spindle assembly during mitosis (Kellogg et al, 1994)
Our findings reveal the critical roles of GCP3 in cell cycle progression, providing insights into the function of its associated complexes, γ-tubulin small complexes (γ-TuSCs) and γ-tubulin ring complexes (γ-TuRCs), in development, and establish a vertebrate model for further study
Consistent with room temperature (RT)-PCR results, whole-mount in situ hybridization (WISH) analyses showed that tubgcp3 mRNA was detected at all developmental stages from cleavage stage to 3 dpf compared with the control group (Figures 1B–J,N,O)
Summary
Centrosome, the major microtubule-organizing center (MTOC) in vertebrate cells, provides a major site for microtubule (MT) nucleation and plays key roles in bipolar spindle assembly during mitosis (Kellogg et al, 1994). A typical centrosome consists of a pair of centrioles surrounded by the pericentriolar matrix (PCM) (Bornens, 2002). Many proteins, including γ-tubulin (GCP1), γ-tubulin complex proteins (GCPs) and a large number of other centrosome-associated proteins, localize to PCM and are involved in the formation of mitotic spindles. The γ-TuSC is a heterotetramer consisting of two copies of γ-tubulin and one copy each of GCP2 and GCP3/Tubgcp. Seven γ-TuSCs with GCP4, GCP5, GCP6 and other accessory proteins assemble into the γ-TuRC, which facilitates MT nucleation by capping the minus ends of MTs and protecting them from depolymerization (Zheng et al, 1995). Centrosomes have critical roles in brain development, and mutations in genes encoding for centrosome-associated proteins have been shown to be genetically linked to neurodevelopmental disorders (Novorol et al, 2013; Chavali et al, 2014; Morris-Rosendahl and Kaindl, 2015; Buchwalter et al, 2016)
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