Fodrin in Centrosomes: Implication of a Role of Fodrin in the Transport of Gamma-Tubulin Complex in Brain

Abstract

Gamma-tubulin is the major protein involved in the nucleation of microtubules from centrosomes in eukaryotic cells. It is present in both cytoplasm and centrosome. However, before centrosome maturation prior to mitosis, gamma-tubulin concentration increases dramatically in the centrosome, the mechanism of which is not known. Earlier it was reported that cytoplasmic gamma-tubulin complex isolated from goat brain contains non-erythroid spectrin/fodrin. The major role of erythroid spectrin is to help in the membrane organisation and integrity. However, fodrin or non-erythroid spectrin has a distinct pattern of localisation in brain cells and evidently some special functions over its erythroid counterpart. In this study, we show that fodrin and γ-tubulin are present together in both the cytoplasm and centrosomes in all brain cells except differentiated neurons and astrocytes. Immunoprecipitation studies in purified centrosomes from brain tissue and brain cell lines confirm that fodrin and γ-tubulin interact with each other in centrosomes. Fodrin dissociates from centrosome just after the onset of mitosis, when the concentration of γ-tubulin attains a maximum at centrosomes. Further it is observed that the interaction between fodrin and γ-tubulin in the centrosome is dependent on actin as depolymerisation of microfilaments stops fodrin localization. Image analysis revealed that γ-tubulin concentration also decreased drastically in the centrosome under this condition. This indicates towards a role of fodrin as a regulatory transporter of γ-tubulin to the centrosomes for normal progression of mitosis.

Binding of alpha‐fodrin to gamma‐tubulin accounts for its role in the inhibition of microtubule nucleation

“…The cell cycle dependent translocation of the c-TuRC to the centrosome in mammalian cells and the peculiar removal of the complex from the centrosome of post mitotic neurons point towards the existence of a stringent mechanism in place to cargo the c-TuRC to and from the MTOC in a temporally regulated fashion. Earlier studies reported by our group showed the colocalization of fodrin and the c-TuRC at the centrosome in brain-derived cells [13,14]. They were also found to be absent in the centrosome in primary neurons.…”

“…Further immunocytochemistry studies in IMR-32 and U251 MG cells also showed the co-localization of c-tubulin with fodrin in centrosome. Moreover, immunoprecipitation experiment using purified centrosomes showed co-immunoprecipitation of fodrin with c-tubulin [14]. Given the apparent co-existence of fodrin and c-tubulin, we performed far western analysis using purified fodrin.…”

“…Further, we found that fodrin co-localized with cytoplasmic as well as centrosomal gamma-tubulin in brain specific cell lines like IMR-32, U-251 MG and primary undifferentiated cultures from brain. However, it was absent in the centrosomes of cervical cancer cell line HeLa or human embryonic kidney cell line HEK-293 [14]. Fodrin is characterized by the presence of spectrin repeats, a repeating unit of about 106 amino acids that forms a helix-loop-helix domain [15].…”

CRISPR/Cas9-mediated integration of large transgene into pig potential safe harbor

“…By using piggybac-mediated transgenesis methods, we generated TG pig lines harboring the target gene in the intron of CEP112 which encodes a centrosomal protein of 112 kDa and is localized around spindle poles, but its structure and function are still unclear (Jakobsen et al . 2011; Shashikala et al . 2013).…”

CRISPR/Cas9-Mediated Integration of Large Transgene into Pig CEP112 Locus

“…Thus, a promising approach that can improve feed efficiency and reduce impacts on the environment is proposed. TG pig lines generated using PiggyBac-mediated transgenesis methods harbor the target gene in the intron of CEP112, which encodes a 112-kD centrosomal protein and is localized around spindle poles; however, the structure and function of the target gene remain unclear (Jakobsen et al 2011;Shashikala et al 2013). The pig lines in our study can efficiently express the foreign gene that can be stably inherited by the offspring, which exhibits normal and healthy performance whether it is heterozygote or homozygote.…”