Generating CRISPR-edited clonal lines of cultured S2 cells.

CRISPR/Cas9 genome editing is a pervasive research tool due to its relative ease of use. However, some systems are not amenable to generating edited clones due to genomic complexity and/or difficulty in establishing clonal lines. For example, Schneider 2 (S2) cells possess a segmental aneuploid genome and are challenging to single-cell select.

Cep104 is a component of the centriole distal tip complex that regulates centriole growth and contributes to Drosophila spermiogenesis.

Proper centrosome number and function relies on the accurate assembly of centrioles, barrel-shaped structures that form the core duplicating elements of the organelle. The growth of centrioles is regulated in a cell cycle-dependent manner; while new daughter centrioles elongate during the S/G2/M phase, mature mother centrioles maintain their length throughout the cell cycle. Centriole length is controlled by the synchronized growth of the microtubules that ensheathe the centriole barrel.

Polo-like kinase 4 homodimerization and condensate formation regulate its own protein levels but are not required for centriole assembly.

Polo-like kinase 4 (Plk4) is the master-regulator of centriole assembly, and cell cycle-dependent regulation of its activity maintains proper centrosome number. During most of the cell cycle, Plk4 levels are nearly undetectable due to its ability to autophosphorylate and trigger its own ubiquitin-mediated degradation. However, during mitotic exit, Plk4 forms a single aggregate on the centriole surface to stimulate centriole duplication.

Balancing the scales: fine-tuning Polo-like kinase 4 to ensure proper centriole duplication.

Polo-like kinase 4 (Plk4) is the master regulator of centriole assembly. Several evolutionarily conserved mechanisms strictly regulate Plk4 abundance and activity to ensure cells maintain a proper number of centrioles. In this issue of , Phan et al.

GLUT3/SLC2A3 Is an Endogenous Marker of Hypoxia in Prostate Cancer Cell Lines and Patient-Derived Xenograft Tumors.

The microenvironment of solid tumors is dynamic and frequently contains pockets of low oxygen levels (hypoxia) surrounded by oxygenated tissue. Indeed, a compromised vasculature is a hallmark of the tumor microenvironment, creating both spatial gradients and temporal variability in oxygen availability. Notably, hypoxia associates with increased metastasis and poor survival in patients.

Centrosome instability: when good centrosomes go bad.

The centrosome is a tiny cytoplasmic organelle that organizes and constructs massive molecular machines to coordinate diverse cellular processes. Due to its many roles during both interphase and mitosis, maintaining centrosome homeostasis is essential to normal health and development. Centrosome instability, divergence from normal centrosome number and structure, is a common pathognomonic cellular state tightly associated with cancers and other genetic diseases.

A molecular mechanism for the procentriole recruitment of Ana2.

During centriole duplication, a preprocentriole forms at a single site on the mother centriole through a process that includes the hierarchical recruitment of a conserved set of proteins, including the Polo-like kinase 4 (Plk4), Ana2/STIL, and the cartwheel protein Sas6. Ana2/STIL is critical for procentriole assembly, and its recruitment is controlled by the kinase activity of Plk4, but how this works remains poorly understood. A structural motif called the G-box in the centriole outer wall protein Sas4 interacts with a short region in the N terminus of Ana2/STIL.

Plk4 Regulates Centriole Asymmetry and Spindle Orientation in Neural Stem Cells.

Defects in mitotic spindle orientation (MSO) disrupt the organization of stem cell niches impacting tissue morphogenesis and homeostasis. Mutations in centrosome genes reduce MSO fidelity, leading to tissue dysplasia and causing several diseases such as microcephaly, dwarfism, and cancer. Whether these mutations perturb spindle orientation solely by affecting astral microtubule nucleation or whether centrosome proteins have more direct functions in regulating MSO is unknown.

An ordered pattern of Ana2 phosphorylation by Plk4 is required for centriole assembly.

Polo-like kinase 4 (Plk4) initiates an early step in centriole assembly by phosphorylating Ana2/STIL, a structural component of the procentriole. Here, we show that Plk4 binding to the central coiled-coil (CC) of Ana2 is a conserved event involving Polo-box 3 and a previously unidentified putative CC located adjacent to the kinase domain. Ana2 is then phosphorylated along its length.

A centrosome interactome provides insight into organelle assembly and reveals a non-duplication role for Plk4.

The centrosome is the major microtubule-organizing centre of many cells, best known for its role in mitotic spindle organization. How the proteins of the centrosome are accurately assembled to carry out its many functions remains poorly understood. The non-membrane-bound nature of the centrosome dictates that protein-protein interactions drive its assembly and functions.