Chromosome misalignment is associated with PLK1 activity at cenexin-positive mitotic centrosomes.

The mitotic kinase, polo-like kinase 1 (PLK1), facilitates the assembly of the two mitotic spindle poles, which are required for the formation of the microtubule-based spindle that ensures appropriate chromosome distribution into the two forming daughter cells. Spindle poles are asymmetric in composition. One spindle pole contains the oldest mitotic centriole, the mother centriole, where the majority of cenexin, the mother centriole appendage protein and PLK1 binding partner, resides. We hypothesized that PLK1 activity is greater at the cenexin-positive older spindle pole. Our studies found that PLK1 asymmetrically localizes between spindle poles under conditions of chromosome misalignment, and chromosomes tend to misalign toward the oldest spindle pole in a cenexin- and PLK1-dependent manner. During chromosome misalignment, PLK1 activity is increased specifically at the oldest spindle pole, and this increase in activity is lost in cenexin-depleted cells. We propose a model where PLK1 activity elevates in response to misaligned chromosomes at the oldest spindle pole during metaphase.

ALS-Linked Mutations Affect UBQLN2 Oligomerization and Phase Separation in a Position- and Amino Acid-Dependent Manner.

Proteasomal shuttle factor UBQLN2 is recruited to stress granules and undergoes liquid-liquid phase separation (LLPS) into protein-containing droplets. Mutations to UBQLN2 have recently been shown to cause dominant X-linked inheritance of amyotrophic lateral sclerosis (ALS) and ALS/dementia. Interestingly, most of these UBQLN2 mutations reside in its proline-rich (Pxx) region, an important modulator of LLPS. Here, we demonstrated that ALS-linked Pxx mutations differentially affect UBQLN2 LLPS, depending on both amino acid substitution and sequence position. Using size-exclusion chromatography, analytical ultracentrifugation, microscopy, and NMR spectroscopy, we determined that those Pxx mutants that enhanced UBQLN2 oligomerization decreased saturation concentrations needed for LLPS and promoted solid-like and viscoelastic morphological changes to UBQLN2 liquid assemblies. Ubiquitin disassembled all LLPS-induced mutant UBQLN2 aggregates. We postulate that the changes in physical properties caused by ALS-linked Pxx mutations modify UBQLN2 behavior in vivo, possibly contributing to aberrant stress granule morphology and dynamics, leading to formation of inclusions, pathological characteristics of ALS.

Regulating a key mitotic regulator, polo-like kinase 1 (PLK1).

During cell division, duplicated genetic material is separated into two distinct daughter cells. This process is essential for initial tissue formation during development and to maintain tissue integrity throughout an organism's lifetime. To ensure the efficacy and efficiency of this process, the cell employs a variety of regulatory and signaling proteins that function as mitotic regulators and checkpoint proteins. One vital mitotic regulator is polo-like kinase 1 (PLK1), a highly conserved member of the polo-like kinase family. Unique from its paralogues, it functions specifically during mitosis as a regulator of cell division. PLK1 is spatially and temporally enriched at three distinct subcellular locales; the mitotic centrosomes, kinetochores, and the cytokinetic midbody. These localization patterns allow PLK1 to phosphorylate specific downstream targets to regulate mitosis. In this review, we will explore how polo-like kinases were originally discovered and diverged into the five paralogues (PLK1-5) in mammals. We will then focus specifically on the most conserved, PLK1, where we will discuss what is known about how its activity is modulated, its role during the cell cycle, and new, innovative tools that have been developed to examine its function and interactions in cells.

Gravin regulates centrosome function through PLK1.

We propose to understand how the mitotic kinase PLK1 drives chromosome segregation errors, with a specific focus on Gravin, a PLK1 scaffold. In both three-dimensional primary prostate cancer cell cultures that are prone to Gravin depletion and Gravin short hairpin RNA (shRNA)-treated cells, an increase in cells containing micronuclei was noted in comparison with controls. To examine whether the loss of Gravin affected PLK1 distribution and activity, we utilized photokinetics and a PLK1 activity biosensor. Gravin depletion resulted in an increased PLK1 mobile fraction, causing the redistribution of active PLK1, which leads to increased defocusing and phosphorylation of the mitotic centrosome protein CEP215 at serine-613. Gravin depletion further led to defects in microtubule renucleation from mitotic centrosomes, decreased kinetochore-fiber integrity, increased incidence of chromosome misalignment, and subsequent formation of micronuclei following mitosis completion. Murine Gravin rescued chromosome misalignment and micronuclei formation, but a mutant Gravin that cannot bind PLK1 did not. These findings suggest that disruption of a Gravin-PLK1 interface leads to inappropriate PLK1 activity contributing to chromosome segregation errors, formation of micronuclei, and subsequent DNA damage.

Evaluating the effectiveness of common disinfectants at preventing the propagation of Mycobacterium spp. isolated from zebrafish.

Mycobacteriosis is a bacterial disease that is common in captive, wild and research fish. There is no one causative agent of mycobacteriosis, as several strains and species of Mycobacterium have been identified in zebrafish. With increased usage and investment in wild-type and mutant zebrafish strains, considerable value is placed on preserving zebrafish health. One control measure used to prevent mycobacterial spread within and between zebrafish facilities is egg disinfection. Here we investigate the effectiveness of three disinfectants [chlorine bleach, hydrogen peroxide, and povidone-iodine (PVPI)] commonly included in egg disinfection protocols for laboratory fish as well as aquaculture fish and compare the knockdown effect of these treatments on Mycobacterium spp. in vitro. Despite current usage, comparison of these disinfection regimes' abilities to prevent mycobacterial growth has not been tested. We found that the germicidal effect of different disinfectants varies by Mycobacterium spp. Hydrogen peroxide was the least effective disinfectant, followed by unbuffered chlorine bleach, which is commonly used to disinfect embryos in zebrafish facilities. Disinfection with 25ppm PVPI for 5min was very effective, and may be an improved alternative to chlorine bleach for embryo disinfection. Results from this study can be utilized by laboratory fish facilities in order to prevent the spread of mycobacteriosis in research fish.