Complete solubilization of mammalian cells in lysates.

In conventional cell lysate protocols, cell debris is typically discarded to obtain a cleaner lysate. However, this approach has limitations, as it may overlook vital cellular components. By discarding cell debris, researchers may inadvertently exclude crucial elements. Retaining all cellular components offers several advantages for studying molecular biology within various cellular compartments. Firstly, it provides a more accurate representation of the cellular environment. Secondly, it enables the study of complex cellular interactions, including those involving cellular structures and signaling pathways associated with debris. This shift in perspective highlights the importance of a holistic approach to lysate preparation. By obtaining lysates that include all cellular components, researchers can gain deeper insights into cellular processes, leading to more accurate data and a better understanding of cellular function and dysfunction. This study aimed to develop a protocol for the preparation of total cell lysates that retain all cellular components, including debris. Our method involves:•A three-step solubilization process using a combination of detergents, saccharides, and chelators, coupled with sonication, in contrast to the classical one-step approach using an all-detergent cocktail.•A comprehensive strategy ensuring the solubilization of all cellular components, providing a more complete lysate for analysis.

Centrosome Movements Are TUBG1-Dependent.

The centrosome of mammalian cells is in constant movement and its motion plays a part in cell differentiation and cell division. The purpose of this study was to establish the involvement of the TUBG meshwork in centrosomal motility. In live cells, we used a monomeric red-fluorescence-protein-tagged centrin 2 gene and a green-fluorescence-protein-tagged TUBG1 gene for labeling the centrosome and the TUBG1 meshwork, respectively. We found that centrosome movements occurred in cellular sites rich in GTPase TUBG1 and single-guide RNA mediated a reduction in the expression of TUBG1, altering the motility pattern of centrosomes. We propose that the TUBG1 meshwork enables the centrosomes to move by providing them with an interacting platform that mediates positional changes. These findings uncover a novel regulatory mechanism that controls the behavior of centrosomes.

Hubbing the Cancer Cell.

Oncogenic transformation drives adaptive changes in a growing tumor that affect the cellular organization of cancerous cells, resulting in the loss of specialized cellular functions in the polarized compartmentalization of cells. The resulting altered metabolic and morphological patterns are used clinically as diagnostic markers. This review recapitulates the known functions of actin, microtubules and the γ-tubulin meshwork in orchestrating cell metabolism and functional cellular asymmetry.

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

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.

The GTPase domain of gamma-tubulin is required for normal mitochondrial function and spatial organization.

In the cell, γ-tubulin establishes a cellular network of threads named the γ-string meshwork. However, the functions of this meshwork remain to be determined. We investigated the traits of the meshwork and show that γ-strings have the ability to connect the cytoplasm and the mitochondrial DNA together. We also show that γ-tubulin has a role in the maintenance of the mitochondrial network and functions as reduced levels of γ-tubulin or impairment of its GTPase domain disrupts the mitochondrial network and alters both their respiratory capacity and the expression of mitochondrial-related genes. By contrast, reduced mitochondrial number or increased protein levels of γ-tubulin DNA-binding domain enhanced the association of γ-tubulin with mitochondria. Our results demonstrate that γ-tubulin is an important mitochondrial structural component that maintains the mitochondrial network, providing mitochondria with a cellular infrastructure. We propose that γ-tubulin provides a cytoskeletal element that gives form to the mitochondrial network.