The histone H3.3 K27M mutation suppresses Ser31phosphorylation and mitotic fidelity, which can directly drive gliomagenesis.

Serine 31 is a phospho-site unique to the histone H3.3 variant; mitotic phospho-Ser31 is restricted to pericentromeric heterochromatin, and disruption of phospho-Ser31 results in chromosome segregation defects and loss of p53-dependant G cell-cycle arrest. Ser31 is proximal to the H3.

Commitment to cytokinetic furrowing requires the coordinate activity of microtubules and Plk1.

At anaphase, spindle microtubules (MTs) position the cleavage furrow and trigger actomyosin assembly by localizing the small GTPase RhoA and the scaffolding protein anillin to a narrow band along the equatorial cortex [1-6]. Using vertebrate somatic cells we examined the temporal control of furrow assembly. Although its positioning commences at anaphase onset, furrow maturation is not complete until ∼10-11 min later.

H3F3A K27M Mutations Drives a Repressive Transcriptome by Modulating Chromatin Accessibility, Independent of H3K27me3 in Diffuse Midline Glioma.

Background: Heterozygous histone H3.3K27M mutation is a primary oncogenic driver of Diffuse Midline Glioma (DMG). H3.

Overcoming translational barriers in H3K27-altered diffuse midline glioma: Increasing the drug-tumor residence time.

Background: H3K27-altered diffuse midline glioma (DMG) is the deadliest pediatric brain tumor; despite intensive research efforts, every clinical trial to date has failed. Is this because we are choosing the wrong drugs? Or are drug delivery and other pharmacokinetic variables at play? We hypothesize that the answer is likely a combination, where optimization may result in a much needed novel therapeutic approach.

Methods: We used in vitro drug screening, patient samples, and shRNA knockdown models to identify an upregulated target in DMG.

StARD9 is a novel lysosomal kinesin required for membrane tubulation, cholesterol transport and Purkinje cell survival.

The pathological accumulation of cholesterol is a signature feature of Niemann-Pick type C (NPC) disease, in which excessive lipid levels induce Purkinje cell death in the cerebellum. NPC1 encodes a lysosomal cholesterol-binding protein, and mutations in NPC1 drive cholesterol accumulation in late endosomes and lysosomes (LE/Ls). However, the fundamental role of NPC proteins in LE/L cholesterol transport remains unclear.

H3K27me3 in Diffuse Midline Glioma and Epithelial Ovarian Cancer: Opposing Epigenetic Changes Leading to the Same Poor Outcomes.

Histone post-translational modifications modulate gene expression through epigenetic gene regulation. The core histone H3 family members, H3.1, H3.

STAT3 is a biologically relevant therapeutic target in H3K27M-mutant diffuse midline glioma.

Background: H3K27M-mutant diffuse midline glioma (DMG) is a lethal brain tumor that usually occurs in children. Despite advances in our understanding of its underlying biology, efficacious therapies are severely lacking.

Methods: We screened a library of drugs either FDA-approved or in clinical trial using a library of patient-derived H3K27M-mutant DMG cell lines with cell viability as the outcome.

Using Microinjection of Mammalian Cultured Cells to Study Cell Division.

The introduction of macromolecules directly into individual cells by microinjection is an important technique for manipulating mitotic cells. mRNA, purified proteins, or concentrated antibodies can all be injected directly into a single cell, and their effects monitored by live-cell imaging. The equipment necessary is relatively simple, and the technique can be easily mastered.

Manipulating cultured mammalian cells for mitosis research.

The study of mitosis has always relied on bulk-preparation biochemistry techniques (Mazia & Dan, 1952), but very early on lent itself to living, single cell microscopic techniques (Inoue, 1953; Taylor, 1959). Here we describe several of the methods used by our lab to study cell division in living cultured cells, including cold-induced mitotic arrest, cold-induced chromosome missegregation, same-cell live and fixed cell imaging, and microinjection of inactivating antibodies. We detail our imaging system based on an upright fluorescent microscope and spinning disk confocal, as well as the customized "HEKS" metal support slide imaging chambers.

Emerging roles for human glycolipid transfer protein superfamily members in the regulation of autophagy, inflammation, and cell death.

Glycolipid transfer proteins (GLTPs) were first identified over three decades ago as ~24kDa, soluble, amphitropic proteins that specifically accelerate the intermembrane transfer of glycolipids. Upon discovery that GLTPs use a unique, all-α-helical, two-layer 'sandwich' architecture (GLTP-fold) to bind glycosphingolipids (GSLs), a new protein superfamily was born. Structure/function studies have provided exquisite insights defining features responsible for lipid headgroup selectivity and hydrophobic 'pocket' adaptability for accommodating hydrocarbon chains of differing length and unsaturation.

The interaction of ceramide 1-phosphate with group IVA cytosolic phospholipase A coordinates acute wound healing and repair.

The sphingolipid ceramide 1-phosphate (C1P) directly binds to and activates group IVA cytosolic phospholipase A (cPLAα) to stimulate the production of eicosanoids. Because eicosanoids are important in wound healing, we examined the repair of skin wounds in knockout (KO) mice lacking cPLAα and in knock-in (KI) mice in which endogenous cPLAα was replaced with a mutant form having an ablated C1P interaction site. Wound closure rate was not affected in the KO or KI mice, but wound maturation was enhanced in the KI mice compared to that in wild-type controls.

Characterization of Novel Murine and Human PDAC Cell Models: Identifying the Role of Intestine Specific Homeobox Gene ISX in Hypoxia and Disease Progression.

Therapy failure and metastasis-associated mortality are stumbling blocks in the management of PDAC in patients. Failure of therapy is associated to intense hypoxic conditions of tumors. To develop effective therapies, a complete understanding of hypoxia-associated changes in genetic landscape of tumors during disease progression is needed.

Microsurgery and microinjection techniques in mitosis research.

The use of microtechnique for studying cell division is well established (Begg & Ellis, 1979; Wadsworth, 1999; Zhang & Nicklas, 1999). The advantage of microinjection in cell division research is the timed delivery of a macromolecules at a particular stage of mitosis (for example, pre- vs postanaphase), which can circumvent the spindle assembly checkpoint (Hinchcliffe et al., 2016).

CPTP: A sphingolipid transfer protein that regulates autophagy and inflammasome activation.

The macroautophagy/autophagy and inflammasome pathways are linked through their roles in innate immunity and chronic inflammatory disease. Ceramide-1-phosphate (C1P) is a bioactive sphingolipid that regulates pro-inflammatory eicosanoid production. Whether C1P also regulates autophagy and inflammasome assembly/activation is not known.

Chromosome missegregation during anaphase triggers p53 cell cycle arrest through histone H3.3 Ser31 phosphorylation.

Maloriented chromosomes can evade the spindle assembly checkpoint and generate aneuploidy, a common feature of tumorigenesis. But chromosome missegregation in non-transformed cells triggers a p53-dependent fail-safe mechanism that blocks proliferation of normal cells that inadvertently become aneuploid. How this fail-safe is triggered is not known.

Centrosomes and the art of mitotic spindle maintenance.

The assembly of a bipolar spindle lies at the heart of mitotic chromosome segregation. In animal somatic cells, the process of spindle assembly involves multiple complex interactions between various cellular compartments, including an emerging antiparallel microtubule network, microtubule-associated motor proteins and spindle assembly factors, the cell's cortex, and the chromosomes themselves. The result is a dynamic structure capable of aligning pairs of sister chromatids, sensing chromosome misalignment, and generating force to segregate the replicated genome into two daughters.

It's all about the pentiums: The use, manipulation, and storage of digital microscopy imaging data for the biological sciences.

Digital microscopy has revolutionized quantitative imaging, with binary-encoded computer files serving to capture and store imaging data sets for analysis. With the ever-present use of computers to generate and store imaging data, it becomes increasingly important to understand how these files are created, and how they can be both used and mis-used. This is a particularly important task for the biologist who may have limited background in computer science.

65,000 shades of grey: use of digital image files in light microscopy.

Computers dominate image capture and analysis in modern light microscopy. The output of an imaging experiment is a binary coded file, called an image file, which contains the spatial, temporal and intensity information present in the sample. Understanding what comprises an image file, and how these files are generated is necessary in order to optimize the use of the digital light microscope.

Post-processing for statistical image analysis in light microscopy.

Image processing of images serves a number of important functions including noise reduction, contrast enhancement, and feature extraction. Whatever the final goal, an understanding of the nature of image acquisition and digitization and subsequent mathematical manipulations of that digitized image is essential. Here we discuss the basic mathematical and statistical processes that are routinely used by microscopists to routinely produce high quality digital images and to extract key features of interest using a variety of extraction and thresholding tools.

Non-vesicular trafficking by a ceramide-1-phosphate transfer protein regulates eicosanoids.

Phosphorylated sphingolipids ceramide-1-phosphate (C1P) and sphingosine-1-phosphate (S1P) have emerged as key regulators of cell growth, survival, migration and inflammation. C1P produced by ceramide kinase is an activator of group IVA cytosolic phospholipase A2α (cPLA2α), the rate-limiting releaser of arachidonic acid used for pro-inflammatory eicosanoid production, which contributes to disease pathogenesis in asthma or airway hyper-responsiveness, cancer, atherosclerosis and thrombosis. To modulate eicosanoid action and avoid the damaging effects of chronic inflammation, cells require efficient targeting, trafficking and presentation of C1P to specific cellular sites.

The centrosome and bipolar spindle assembly: does one have anything to do with the other?

In vertebrate somatic cells the centrosome functions as the major microtubule-organizing center (MTOC), which splits and separates to form the poles of the mitotic spindle. However, the role of the centriole-containing centrosome in the formation of bipolar mitotic spindles continues to be controversial. Cells normally containing centrosomes are still able to build bipolar spindles after their centrioles have been removed or ablated.

Zwint-1 is a novel Aurora B substrate required for the assembly of a dynein-binding platform on kinetochores.

Aurora B (AurB) is a mitotic kinase responsible for multiple aspects of mitotic progression, including assembly of the outer kinetochore. Cytoplasmic dynein is an abundant kinetochore protein whose recruitment to kinetochores requires phosphorylation. To assess whether AurB regulates recruitment of dynein to kinetochores, we inhibited AurB using ZM447439 or a kinase-dead AurB construct.

Polo-like kinase1 is required for recruitment of dynein to kinetochores during mitosis.

Kinetochore dynein has been implicated in microtubule capture, correcting inappropriate microtubule attachments, chromosome movement, and checkpoint silencing. It remains unclear how dynein coordinates this diverse set of functions. Phosphorylation is responsible for some dynein heterogeneity (Whyte, J.

Amphiastral mitotic spindle assembly in vertebrate cells lacking centrosomes.

The role of centrosomes and centrioles during mitotic spindle assembly in vertebrates remains controversial. In cell-free extracts and experimentally derived acentrosomal cells, randomly oriented microtubules (MTs) self-organize around mitotic chromosomes and assemble anastral spindles. However, vertebrate somatic cells normally assemble a connected pair of polarized, astral MT arrays--termed an amphiaster ("a star on both sides")--that is formed by the splitting and separation of the microtubule-organizing center (MTOC) well before nuclear envelope breakdown (NEB).

Centrosome biogenesis continues in the absence of microtubules during prolonged S-phase arrest.

When CHO cells are arrested in S-phase, they undergo repeated rounds of centrosome duplication without cell-cycle progression. While the increase is slow and asynchronous, the number of centrosomes in these cells does rise with time. To investigate mechanisms controlling this duplication, we have arrested CHO cells in S-phase for up to 72 h, and coordinately inhibited new centriole formation by treatment with the microtubule poison colcemid.