Search

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. Here, we demonstrate that NPC1 mutations impair the projection of cholesterol-containing membrane tubules from the surface of LE/Ls. A proteomic survey of purified LE/Ls identified StARD9 as a novel lysosomal kinesin responsible for LE/L tubulation. StARD9 contains an N-terminal kinesin domain, a C-terminal StART domain, and a dileucine signal shared with other lysosome-associated membrane proteins. Depletion of StARD9 disrupts LE/L tubulation, paralyzes bidirectional LE/L motility and induces accumulation of cholesterol in LE/Ls. Finally, a novel StARD9 knock-out mouse recapitulates the progressive loss of Purkinje cells in the cerebellum. Together, these studies identify StARD9 as a microtubule motor protein responsible for LE/L tubulation and provide support for a novel model of LE/L cholesterol transport that becomes impaired in NPC disease.

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. A single small molecule protein kinase inhibitor with translational potential was selected for systemic and direct, loco-regional delivery to patient-derived xenografts (PDX) and genetically engineered mouse models (GEMM). Pharmacokinetic studies were conducted in non-tumor bearing rats. Results Aurora kinase (AK) inhibitors demonstrated strong antitumor effects in DMG drug screens. Additional in vitro studies corroborated the importance of AK to DMG survival. Systemic delivery of alisertib showed promise in subcutaneous PDX but not intracranial GEMM and PDX models. Repeated loco-regional drug administration into the tumor through convection-enhanced delivery (CED) was equally inefficacious, and pharmacokinetic studies revealed rapid clearance of alisertib from the brain. In an effort to increase the drug to tumor residence time, continuous CED over 7 days improved drug retention in the rodent brainstem and significantly extended survival in both orthotopic PDXs and GEMMs. Conclusions These studies provide evidence for increasing drug-tumor residence time of promising targeted therapies via extended CED as a valuable treatment strategy for DMG.

During the cell cycle, differential phosphorylation of select histone H3 serine/threonine residues regulates chromatin structure, necessary for both dynamic transcriptional control and proper chromosome segregation1-2. Histone H3.3 contains a highly conserved serine residue (Ser31) within its N-terminal tail that is unique to this variant. During interphase phosphorylation of Ser31 amplifies stimulation-induced transcription and is required for early metazoan development3-6. During mitosis Ser31 phosphorylation at the pericentromere supports proper chromosome segregation, albeit by unknown mechanisms7-10. H3.3 Ser31 is flanked by mutational sites that drive several human cancers, including pediatric gliomas5-8. This is typified by the H3.3K27M mutation found in ∼80% of diffuse midline gliomas, which undergo epigenetic reprogramming in proliferative cells coordinate with loss of global H3 lysine 27 trimethylation (H3K27Me3)11-14. However, whether the K27M mutation influences the neighboring Ser31 phosphorylation and whether disrupting Ser31 phosphorylation plays a distinct role in driving gliomagenesis has not been tested. Here we show that H3.3K27M mutant cells have reduced capacity for H3.3 Ser31 phosphorylation at the mitotic pericentromere, increased rates of chromosome missegregation, and impaired G1 checkpoint responses to chromosome instability. CRISPR-reversion of K27M to wild-type restores phospho-Ser31 levels and suppresses chromosome segregation defects. CRISPR editing to introduce a non-phosphorylatable H3.3S31A alone is sufficient to increase the frequency of chromosome missegregations. Finally, expression of H3.3S31A in a PDGFβ-driven RCAS/TVA mouse model is sufficient to drive high grade gliomagenesis, generating diffuse tumors morphologically indistinguishable from those generated by H3.3K27M expression. Importantly, this occurs without the loss of H3K27 triple methylation that is the hallmark of K27M tumors. Our results reveal that the H3.3 K27M mutation alters the neighboring Ser31 phosphorylation, and loss of proper H3.3 Ser31 phosphorylation contributes to the formation of diffuse midline gliomas.

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. Results were validated for clinical relevance and mechanistic importance using patient specimens from biopsy and autopsy, patient-derived cell lines, inhibition by gene knockdown and small molecule inhibitors, and patient-derived xenografts. Results Kinase inhibitors were highly toxic to H3K27M-mutant DMG cells. Within this class, STAT3 inhibitors demonstrated robust cytotoxic activity in vitro. Mechanistic analyses revealed one form of activated STAT3, phospho-tyrosine- 705 STAT3 (pSTAT3), was selectively upregulated in H3K27M-mutant cell lines and clinical specimens. STAT3 inhibition by CRISPR/Cas9 knockout, shRNA or small molecule inhibition reduced cell viability in vitro, and partially restored expression of the polycomb repressive mark H3K27me3, which is classically lost in H3K27M-mutant DMG. Putative STAT3-regulated genes were enriched in an H3K27M-knockout DMG cell line, indicating relative gain of STAT3 signaling in K27M-mutant cells. Treatment of patient-derived intracranial xenografts with WP1066, a STAT3 pathway inhibitor currently in clinical use for pediatric brain tumors, resulted in stasis of tumor growth, and increased overall survival. Finally, pSTAT3(Y705) was detected in circulating plasma extracellular vesicles of patients with H3K27M-mutant DMG. Conclusions STAT3 is a biologically relevant therapeutic target in H3K27M-mutant DMG. STAT3 inhibition should be considered in future clinical trials.

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. Here we describe our microinjection setup, how to microinject cultured mammalian cells in mitosis, and how to analyze those cells by same-cell live and fixed imaging.

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. Because artificially immortalized cell lines do not rightly represent the disease progression, studying genetics of tumors in spontaneous models is warranted. In the current study, we generated a spectrum of spontaneous human (UM-PDC1; UM-PDC2) and murine (HI-PanL, HI-PancI, HI-PanM) models representing localized, invasive, and metastatic PDAC from a patient and transgenic mice (K-rasG12D/Pdxcre/Ink4a/p16-/). These spontaneous models grow vigorously under hypoxia and exhibit activated K-ras signaling, progressive loss of PTEN, and tumorigenicity in vivo. Whereas UM-PDC1 form localized tumors, the UM-PDC2 metastasize to lungs in mice. In an order of progression, these models exhibit genomic instability marked by gross chromosomal rearrangements, centrosome-number variations, Aurora-kinase/H2AX colocalization, loss of primary cilia, and α-tubulin acetylation. The RNA sequencing of hypoxic models followed by qRT-PCR validation and gene-set enrichment identified Intestine-Specific Homeobox factor (ISX)–driven molecular pathway as an indicator PDAC aggressivness. TCGA-PAAD clinical data analysis showed high ISX expression correlation to poor survival of PDAC patients, particularly women. The functional studies showed ISX as a regulator of i) invasiveness and migratory potential and ii) VEGF, MMP2, and NFκB activation in PDAC cells. We suggest that ISX is a potential druggable target and newly developed spontaneous cell models are valuable tools for studying mechanism and testing therapies for PDAC.

Histone H3.3 G34R/V mutations are drivers of pediatric high-grade glioma (pHGG). However, the mechanism(s) responsible for G34R/V induced tumorigenesis are unclear. We observed that pHGG cells with H3.3 G34 mutations have significantly reduced phosphorylation at H3.3 S31 as compared to H3.3 WT cell lines. And, in vitro, G34 mutation completely blocks H3.3 S31 phosphorylation by Chk1 – the kinase responsible for S31 phosphorylation. During early mitosis, phospho-S31 is enriched in the chromosomal pericentromere; which plays a vital structural role in chromosome segregation. Over-expression of GFP-H3.3 G34R or non-phosphorylatable GFP-H3.3 S31A in chromosomally stable cell lines results in a significant increase in chromosome mis-segregations. Likewise, H3.3 G34V pHGG derived cells showed a higher frequency of mis-segregation than H3.3 WT cells. During cell division, phospho-S31 is lost in late anaphase. However, when chromosome missegregation occurs, phospho-S31 spreads to the chromosome arms where it stimulates p53 accumulation in G1 – thus acting as a sensor to suppress the proliferation of aneuploid cells. Here we show that cells expressing mutant G34 fail to arrest following mis-segregation, despite having WT p53. Taken together this work demonstrates that the H3.3 G34R/V mutations are sufficient to transform normal, diploid cells into proliferative, chromosomally instable cells. To determine if this process contributes to tumorigenesis, we expressed WT H3.3, H3.3-G34R or H3.3-S31A in a mouse model of pHGG. H3.3 WT controls developed low-grade tumors and all survived 80 days. Mice with H3.3-G34R or H3.3-S31A developed low- and high-grade tumors. And 78% and 50% of H3.3-G34R and H3.3-S31A mice, respectively, survived to 80 days. Our work suggests that H3.3 G34R/V drives chromosomal instability through the suppression of H3.3 S31 phosphorylation AND that chromosomal instability is a contributing driver of glioma formation in G34 mutant tumors.

Pediatric glioblastoma and diffuse intrinsic pontine glioma are high-grade gliomas of children (pHGG) with a median overall survival of under 15 months and among the most lethal cancers. Mutations in histone H3.3 and H3.1 occur as an early event in pHGG. H3.3G34R/V-mutations occur in pHGG of cerebral hemispheres, and H3.3K27M mutations occur in midline pHGGs. Post-translational histone modifications (PTMs) serve to regulate gene expression by relaxing or compacting chromatin and by recruiting proteins, with subsequent silencing or activating effects. H3.3 Serine 31 (S31) shows reduced phosphorylation during mitosis in H3.3G34R/V and H3.3K27M mutant cell. Phosphorylation at S31 is restored in wildtype H3.3K27 CRISPR revertants. Serine to alanine (A) mutant H3.3 S31A are nonphosphorylatable in vitro. To study the influence of histone mutations and the role of altered PTM and including the loss of methylation and phosphorylation on tumorigenesis, we have developed an innovative model based on the RCAS/N-TVA mouse model. In this system, the expression of an oncogenic driver is linked to mutant histone expression using a self-cleaving peptide, and tumors develop following viral delivery to neural stem cells in newborn mice. This approach is necessary, as otherwise, clonal selection could prevent tumors from forming with mutations detrimental to growth. To establish the model, N-TVA mice were injected with RCAS H3.3K27M-P2A-PDGFB, RCAS H3.3G34R-P2A-PDGFB, or H3.3WT-P2A-PDGFB. The mean survival of mice injected with H3.3K27M and H3.3S31A was 81 and 68 days, respectively, and 100% of S31A mice developed HGG. In contrast, H3.3WT caused only low-grade tumors in 46% of the mice, and all mice survived until 100 days. In ongoing experiments with H3.3G34R, 23% of mice succumb to tumors by 80 days. These results provide mechanistic insights into the early establishment of pHGGs and established a new mouse model to study the role of histone mutation and PTMs in tumor development.

Adherent cells cultured on flat, homogeneous surfaces typically maintain an intact cell body with a polygonal or fan shape, despite active migration and strong mechanical interactions with the substratum. We hypothesized that, in addition to the constraint of the surface membrane, an active mechanism may be involved in maintaining the shape and integrity of the cell body particularly where cells encounter complex topographic patterns of guidance cues. To detect if there is a mechanism that constrains cell shape, we plated NIH 3T3 fibroblasts on ring-patterned substrata 8–17.5 microns in width and 53–133 microns in outer diameter. Untreated cells have a limited angular span, encompassing an average of 108 degrees around the ring, even though these cells were able to cover a much larger surface when plated on flat surfaces of the same material. Treatment of 3T3 cells with a myosin II inhibitor, blebbistatin, induced a striking increase in the bending ability, causing cells to cover more than 60% of the ring. Inhibition of the Rho-dependent kinase with Y-27632 caused a similar but smaller increase in the angular span. Our results suggest that cell shape is controlled not only by the passive constraint of the surface membrane but also by an active mechanism driven by myosin II-mediated contractility under the regulation of Rho-dependent kinase. The inward surface tension-like forces allow the cell to maintain its integrity while navigating through complex physiological environments.

Pediatric midline gliomas – including DIPG – are lethal brain tumors in children, with poor prognosis and limited treatment options that provide only short-term benefits. The majority have a lysine-to-methionine substitution at residue 27 (H3K27M) in genes expressing histone H3 – predominantly in the H3.3 variant. This causes a global reduction in H3 Lys27 tri-methylation (H3K27Me3), comprehensive epigenetic reprogramming, and is a key driver in gliomagenesis. We show that the H3.3K27M mutation also induces chromosome segregation defects, which in high-grade tumors, results in extensive copy number alterations (CNAs). Ser31 is one of five amino acid substitutions differentiating H3.3 from canonical H3.1. Mitotic phosphorylation of H3.3 Ser31 by Chk1 kinase is restricted to pericentromeric heterochromatin, where it plays a role in chromosome segregation. We show that the K27M mutation affects neighboring Ser31 phosphorylation and pericentromeric heterochromatin organization. We demonstrate that (i) H3.3 K27M protein is defective for Ser31 phosphorylation by Chk1 kinase in vitro; (ii) DIPG cell lines have significantly decreased mitotic Ser31 phosphorylation, and are chromosomally unstable; and (iii) CRISPR-reversion of H3.3K27M to Lys27 restores phospho-Ser31 (and Lys27 tri-methylation) and significantly decreases chromosome instability. Expression of H3.3K27M or non-phosphorylatable H3.3S31A mutants in WT cells results in chromosome missegregation; this is suppressed by co-expression of phospho-mimetic H3.3K27M/S31E. In normal cells, chromosome missegregation stimulates p53-dependent cell cycle arrest in G1 to prevent the proliferation of aneuploid daughters. However, cells expressing H3.3 K27M or S31A failed to arrest following missegregation - despite having WT p53. Finally, in a novel mouse model of glioma, mean survival of mice with tumors induced with H3.3K27M and H3.3S31A was 81 and 68 days: 100% of H3.3S31A mice developed high-grade tumors. H3.3 WT controls developed only low-grade tumors and all survived 100 days. H3.3S31A is WT for Lys27 tri-methylation and thus, loss of Ser31 phosphorylation alone is oncogenic.

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.

The sphingolipid ceramide-1-phosphate (C1P) directly binds to and activates group IVA cytosolic phospholipase A(2) (cPLA(2)α) 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 cPLA(2)α and in knock-in (KI) mice in which endogenous cPLA(2)α 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. Wounds in KI mice displayed increased infiltration of dermal fibroblasts into the wound environment, increased wound tensile strength, and a higher ratio of type I:type III collagen. In vitro, primary dermal fibroblasts (pDFs) from KI mice showed substantially increased collagen deposition and migration velocity compared to pDFs from wild-type and KO mice. Additionally, KI mice showed an altered eicosanoid profile of reduced proinflammatory prostaglandins (PGE(2) and TXB(2)) and an increased abundance of certain hydroxyeicosatetraenoic acid (HETE) species. Specifically, an increase in 5-HETE enhanced dermal fibroblast migration and collagen deposition. This gain-of-function role for the mutant cPLA(2)α was also linked to the relocalization of cPLA(2)α and 5-HETE biosynthetic enzymes to the cytoplasm and cytoplasmic vesicles. These findings demonstrate the regulation of key wound-healing mechanisms in vivo by a defined protein-lipid interaction and provide insights into the roles that cPLA(2)α and eicosanoids play in orchestrating wound repair.

The sphingolipid ceramide 1-phosphate (C1P) directly binds to and activates group IVA cytosolic phospholipase A2 (cPLA2α) 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 cPLA2α and in knock-in (KI) mice in which endogenous cPLA2α 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. Wounds in KI mice displayed increased infiltration of dermal fibroblasts into the wound environment, increased wound tensile strength, and a higher ratio of type I:type III collagen. In vitro, primary dermal fibroblasts (pDFs) from KI mice showed substantially increased collagen deposition and migration velocity compared to pDFs from wild-type and KO mice. KI mice also showed an altered eicosanoid profile of reduced proinflammatory prostaglandins (PGE2 and TXB2) and an increased abundance of certain hydroxyeicosatetraenoic acid (HETE) species. Specifically, an increase in 5-HETE enhanced dermal fibroblast migration and collagen deposition. This gain-of-function role for the mutant cPLA2α was also linked to the relocalization of cPLA2α and 5-HETE biosynthetic enzymes to the cytoplasm and cytoplasmic vesicles. These findings demonstrate the regulation of key wound-healing mechanisms in vivo by a defined protein-lipid interaction and provide insights into the roles that cPLA2α and eicosanoids play in orchestrating wound repair.

BACKGROUND: Heterozygous mutations in Histone H3.3 (K27M, G34R/V) occur in 70–80% of pediatric high-grade gliomas and 78% of diffuse intrinsic pontine gliomas (DIPG). These mutations are also found in low-grade gliomas that progress to higher grade tumors. Epigenetic dysregulation resulting from altered methylation at or near the mutated sites is believed to drive malignant transformation H3 differs from H3.1 by only 5 amino acids, which includes a unique phosphorylation site, Serine 31. S31 is phosphorylated by Chk1 kinase at pericentromeric heterochromatin in mitosis and on the arms of missegregated chromosomes. Masking S31 phosphorylation by antibody injection or using a non-phosphorylatable S31A substitution prevents cell cycle arrest in response to chromosome missegregation. OBJECTIVE: To determine if H3.3 K27M or G34R/V mutations alter the regulation of S31 phosphorylation to promote chromosome missegregation, aneuploidy, and tumorigenesis. METHODS: Live cell imaging and an established mouse model of glioma. RESULTS: DIPG cells with the K27M mutations show decreased mitotic Ser31 phosphorylation and increased chromosome missegregation. In both model cell lines and normal astrocytes, K27M and G34R mutations abolish S31 phosphorylation. Expression of S31A, K27M, or G34R mutants results in increased chromosome missegregation and aneuploidy, whereas cells expressing phosphomimetic double mutants (K27M/S31E or G34R/S31E) divide normally. Using the RCAS/TVA mouse model, H3.3WT, K27M, and S31A constructs were introduced into the brains of newborn mice along with mouse BRAF(V600E). 0/13 tumors developed in the BRAF(V600E) + H3.3WT cohort; however, 80% (16/20) of H3.3K27M + BRAF(V600E) and 70% (7/10) H3.3S31A + BRAF(V600E) cohorts developed high-grade gliomas within 100 days. CONCLUSION: A single heterozygous H3.3 mutation is sufficient to generate aneuploid cells, allows them to reenter the cell cycle and to promote tumorigenesis. Restoring cell cycle checkpoint activity by modulating S31 phosphorylation may be a rational treatment for DIPG or to prevent the progression of low-grade gliomas with H3.3 mutations.

The histone H3.3 G34R/V mutations are known drivers of high-grade pediatric glioma (pHGG). However, the mechanism(s) for H3.3 G34R/V induced tumor formation are unclear. Chk1 phosphorylates H3.3 S31 at the pericentromere during early mitosis, suggesting a novel mitotic function. We observed that H3.3 G34 mutant pHGG cells have reduced mitotic H3.3 S31 phosphorylation compare to WT H3.3 cell lines. The H3.3 G34R mutation reduced Chk1 phosphorylation at S31 by >90% in an in vitro kinase assay. Overexpression of either H3.3 G34R or non-phosphorylatable S31A in H3.3 WT, diploid cells led to a significant increase in chromosome mis-segregations. Likewise, H3.3 G34 mutant pHGG cells have significantly elevated rates of mis-segregation as compare to H3.3 WT pHGG cells. During normal cell division, phospho-S31 is lost in late anaphase. However, when chromosome missegregation occurs, phospho-S31 spreads and stimulates p53 accumulation in G1 – thus suppressing aneuploid cell proliferation. Here we show that cells expressing mutant G34 fail to arrest following mis-segregation, despite having WT p53. These studies demonstrate that the H3.3 G34R/V mutations are sufficient to transform normal, diploid cells into proliferative, chromosomally instable cells. To determine if this process contributes to tumorigenesis, we used the RCAS/TVA mouse model to overexpress H3.3 WT, G34R, or S31A in the glial precursor cells of mice pups. Over 100 days, S31A and G34R mice had drastically reduced survival (averaging 77, 81, and 100 days for S31A, G34R, and WT mice). Furthermore, most G34R and S31A mice developed HGG, while H3.3 WT mice remained tumor-free and did not develop high-grade tumors. Our work strongly indicates that a major factor in H3.3 G34R pHGG formation is the induction of chromosomal instability – which occurs directly through the suppression of H3.3 S31 phosphorylation.

Diffuse midline gliomas with the H3.3 K27M mutation are lethal brain tumors in children. H3 K27M causes global loss of Lys27 triple methylation (Lys27me3), inducing epigenetic reprograming. Here we show that H3.3 K27M also causes decreased H3.3 Ser31 phosphorylation on mitotic chromosomes. We show that H3.3 K27M DIPG cells have reduced pericentromeric phospho-Ser31 and increased rates of chromosome missegregation compared to normal, diploid human cells. CRISPR-editing K27M to M27K restored phospho-Ser31 to WT levels and dramatically decreased the rate of chromosome missegregation. We confirm that Chk1 is the H3.3 Ser31 kinase: K27M mutant H3.3 protein exhibits ~60% reduced Chk1 phosphorylation of Ser31 in vitro. Chk1 knockdown completely abolishes phospho-Ser31 in cells and these have increased rates of chromosome missegregation. In normal, diploid cells, expression of K27M or an S31A non-phosphorylatable mutant increased chromosome missegregation; this is suppressed by expressing a phosphomimetic double mutant (K27M/S31E) that restores phospho-Ser31. WT cells arrest following chromosome missegregation. However, cells expressing H3.3 K27M or S31A fail to arrest - despite having WT p53. Finally, we expressed H3F3AS31A and PDGFb in an RCAS/TVA mouse model of DIPG and ~80% developed diffuse high-grade brain tumors and show significantly decreased survival. Our results suggest that loss of phospho-Ser31 alone is oncogenic because H3.3 S31A-expressing cells are WT for K27me3. Our results demonstrate that H3.3 K27M inhibits Ser31 phosphorylation both in vitro and in vivo, leading to both chromosome missegregation and loss of subsequent G1 arrest – thus creating diffuse midline gliomas with both dynamic, complex karyotypes and epigenetic reprogramming.

Diffuse midline glioma (DMG) is a lethal brain tumor that typically occurs in children. Numerous studies have demonstrated the central role of the H3K27M mutation and secondary loss of H3K27 trimethylation (H3K27me3) in DMG tumorigenesis. Understanding how the H3K27M mutation alters the epigenetic landscape of the cell is necessary for revealing molecular targets that are critical to tumorigenesis. To investigate the epigenetic effects of H3K27M mutation in DMG, we developed revertant DMG cell lines with the mutant methionine residue reverted to wildtype (i.e., M27K). Revertant cells were analyzed for epigenetic changes and phenotypic differences in vitro and in vivo. H3M27K DMG cells grew in culture but displayed diminished proliferative capacity. H3M27K cells demonstrated total loss of H3K27M expression and restored trimethylation of H3K27 and H3K4. Furthermore, consistent with the hypothesis that the H3K27M mutation impacts H3 phosphorylation via expression of Aurora Kinase during mitosis, H3M27K cells demonstrated reduced expression of both Aurora Kinase A and phosphorylation of H3 serine residues 10 and 28. In line with the critical role of H3S10 phosphorylation in chromatin segregation, H3M27K cells also demonstrated restored chromosome segregation compared to H3K27M cells. In vivo data will be discussed. Revertance of the H3K27M mutation reduces tumorigenesis in DMG tumors. Isogenic H3M27K cells display reversal of key epigenetic changes associated with oncogenesis in DMG. The revertant H3M27K DMG model is a useful tool to investigate the downstream epigenetic reprogramming specific to H3K27M mutation in these tumors.

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. In humans, evolutionarily-modified GLTP-folds have been identified with altered sphingolipid specificity, e. g. ceramide-1-phosphate transfer protein (CPTP), phosphatidylinositol 4-phosphate adaptor protein-2 (FAPP2) which harbors a GLTP-domain and GLTPD2. Despite the wealth of structural data (> 40 Protein Data Bank deposits), insights into the in vivo functional roles of GLTP superfamily members have emerged slowly. In this review, recent advances are presented and discussed implicating human GLTP superfamily members as important regulators of: i) pro-inflammatory eicosanoid production associated with Group-IV cytoplasmic phospholipase A(2); ii) autophagy and inflammasome assembly that drive surveillance cell release of interleukin-1β and interleukin-18 inflammatory cytokines; iii) cell cycle arrest and necroptosis induction in certain colon cancer cell lines. The effects exerted by GLTP superfamily members appear linked to their ability to regulate sphingolipid homeostasis by acting in either transporter and/or sensor capacities. These timely findings are opening new avenues for future cross-disciplinary, translational medical research involving GLTP-fold proteins in human health and disease. Such avenues include targeted regulation of specific GLTP superfamily members to alter sphingolipid levels as a therapeutic means for combating viral infection, neurodegenerative conditions and circumventing chemo-resistance during cancer treatment.

The use of microtechnique for studying cell division is well established (BeggEllis, 1979; Wadsworth, 1999; ZhangNicklas, 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). Micromanipulation can be used to remove whole organelles, such as the centrosome or nucleus and examine the effects on cell division (Hinchcliffe et al., 2001; Hornick et al., 2011). The focus of this chapter is on methods for microinjection and micromanipulation of cultured mammalian cells. We describe pulling and shaping microneedles, as well as the imaging chambers we use. We also provide information on cell culture conditions, and imaging techniques used for our long-term observation studies, which allow cells to be followed on the order of several days.

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. Here we show that CPTP (a protein that traffics C1P from its site of phosphorylation in the trans-Golgi to target membranes) regulates both autophagy and inflammasome activation. In human epithelial cells, knockdown of CPTP (but not GLTP [glycolipid transfer protein]) or expression of C1P binding-site point mutants, stimulated an 8- to 10-fold increase in autophagosomes and altered endogenous LC3-II and SQSTM1/p62 protein expression levels. CPTP depletion-induced autophagy elevated early markers of autophagosome formation (Golgi-derived ATG9A-vesicles, WIPI1), required key phagophore assembly and elongation factors (ATG5, ATG7, ULK1), and suppressed MTOR phosphorylation and that of its downstream target, RPS6KB1/p70S6K. Wild-type CPTP overexpression exerted a protective effect against starvation-induced autophagy. In THP-1 macrophage-like surveillance cells, CPTP knockdown induced not only autophagy but also elevated CASP1/caspase-1 levels, and strongly increased IL1B/interleukin-1β and IL18 release via a NLRP3 (but not NLRC4) inflammasome-based mechanism, while only moderately increasing inflammatory (pyroptotic) cell death. Inflammasome assembly and activation stimulated by CPTP depletion were autophagy dependent. Elevation of intracellular C1P by exogenous C1P treatment (instead of CPTP inhibition) also induced autophagy and IL1B release. Our findings identify human CPTP as an endogenous regulator of early-stage autophagosome assembly and inflammasome-driven, pro-inflammatory cytokine generation and release.

Diffuse intrinsic pontine glioma (DIPG) carries a signature mutation in histone H3.3 (K27M) that induces epigenetic reprogramming via suppressed Lys27 triple methylation (K27me3). H3.3 has a Ser at position 31, which is adjacent to the K27M DIPG mutation. H3.3 Ser31 is phosphorylated beginning in prophase. This phosphorylation is restricted to pericentromeric heterochromatin and becomes dephosphorylated in anaphase. Surprisingly, chromosome missegregation triggers Ser31 hyperphosphorylation on the lagging chromosome, and masking phosphoS31 prevents p53 accumulation; part of the G1 checkpoint response to aneuploidy. Whether the K27M mutation influences Ser31 phosphorylation, chromosome segregation or G1 checkpoint control is unknown. We show that K27M DIPG cells have reduced pericentromeric phosphoS31 and increased chromosome instability (CIN) compared to normal, diploid human cells. CRISPR-editing of K27M to M27K restored phosphoS31 to WT levels and dramatically decreased the rate of CIN. We also demonstrate that Chk1 is the H3.3 Ser31 kinase. S31A abolishes Ser31 phosphorylation by Chk1, while K27M reduces it by ~60% in vitro. Chk1 knockdown abolishes phosphoS31 in normal, diploid mitotic cells, and induces CIN. In normal, diploid cells, expression of S31A or K27M mutations increased chromosome missegregation, whereas cells expressing a phosphomimetic double mutant (K27M/S31E) divide normally. WT cells arrest following chromosome missegregation. Yet, normal cells expressing H3.3 K27M or S31A do not arrest - despite having wild-type p53. Finally, using RCAS/TVA, we expressed H3F3AS31A in the brains of BRAFV600E mice, and ~50% developed tumors, suggesting that loss of phosphoS31 alone is oncogenic. Importantly, H3.3 S31A-expressing cells are WT for K27me3. Our results demonstrate that expressing H3.3 K27M reduces the population of H3.3 that is phosphorylatable at Ser31, leading to chromosome missegregation and preventing G1 arrest – thus creating proliferating CIN cells. We propose that a single amino acid substitution drives oncogenesis of DIPG, at least impart, by triggering the evolution of complex karyotypes.

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). Micromanipulation can be used to remove whole organelles, such as the centrosome or nucleus and examine the effects on cell division (Hinchcliffe et al., 2001; Hornick et al., 2011). The focus of this chapter is on methods for microinjection and micromanipulation of cultured mammalian cells. We describe pulling and shaping microneedles, as well as the imaging chambers we use. We also provide information on cell culture conditions, and imaging techniques used for our long-term observation studies, which allow cells to be followed on the order of several days.

Introduction: Adeno-associated virus (AAV) is a small virus of the parvovirus family and is widely used in clinical-stage gene therapy because of its low toxicity and limited side effects. There are a number of naturally occurring serotypes with different tissue tropism and further engineering of the viral capsid can increase the vectors suitability for use as a cancer vaccine. We recently showed that AAV serotype 6 (AAV6) successfully delivers a transgene to dendritic cells (DC) in vitro. In the present study we analyze several rationally designed AAV6 mutants to more efficiently target DC and further increase an antigen-specific immune response. Methods: AAV6 mutants encoding ovalbumin, tyrosinase, or GFP were developed and produced in the lab by standard procedure. Vaccination was performed as a single intramuscular (i.m.) injection in C57BL/6 mice. The immune response was measured against both ovalbumin (Ova) as a model antigen and tyrosinase (Tyr) as an endogenous antigen. The Ova-specific CD8 T-cells were identified in peripheral blood and splenocytes by binding epitope-specific tetramers. Activity of antigen-specific CD8 cells was estimated by IFNy production measured by ELISpot, and by in vivo and in vitro killing assays. A humoral response was analyzed by the presence of antigen-specific antibodies in mouse serum measured by ELISA or by Western Blotting. Vaccination with GFP was used to show direct antigen expression in DC. Briefly, 3 days after i.m. injection DC from drained lymph nodes were isolated by FACS (CD11c+/MHC II+) and the presence of GFP expression was analyzed by wide-field fluorescence microscopy. Results: Several significant observations were made: (i) optimized AAV6 directly targets DC in vivo as evidenced by the presence of GFP positive DC in mouse drained lymph nodes after i.m. injection with AAV6-GFP; (ii) AAV6 vectors induced a strong immune response not only against a strong immunogen such as Ova but also against a weak endogenous antigen such as Tyr. (iii) Transgene-specific cytotoxic CD8 T- cells showed superior killing capabilities against cancer cells in vitro and in cytotoxic assay in vivo. (iv) AAV6 based vaccine generates high transgene-specific antibodies titers detectable starting 3 weeks after injection. Our data allows us to choose the AAV6 mutant with superior capacity to induce an immune response against a transgene. As a future direction we will optimize the expression cassette to directly rout an antigen into the MHC class II processing and presentation pathway which is known to dramatically increase vaccine potency. The combination of optimized AAV capsid and expression cassette will be used to confirm an ability of our vaccine approach to reduce tumor growth and extend survival in a murine tumor model. In addition, our technology is highly compatible with, and complementary to current cancer treatments, including other immunotherapy approaches such as checkpoint (PD-1/PD-L1) inhibitors. Citation Format: Karina Krotova, Andrew Day, Edward Hinchcliffe, George Aslanidi. Bioengineering AAV6 vector-based vaccine for cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1467.

SUMMARY 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. Here we discuss some of the basic aspects of digital data storage and use, including file types, storage media, and the choice between commercial and open-source software. Often, open-source software is written by a user or group of users, and then distributed to the scientific community at large. These can be important tools, but there are some hidden costs to this freeware that we will discuss. We will also compare open-source software to commercial imaging software, which is often written for use by non-computer scientists. Mol. Reprod. Dev. 82: 508–517, 2015. © 2014 Wiley Periodicals, Inc.

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. In naturally occurring cellular systems that lack centrioles - such as plant cells and many oocytes - bipolar spindles form in the complete absence of canonical centrosomes. These observations have led to the notion that centrosomes play no role during mitosis. However, recent work has re-examined spindle assembly in the absence of centrosomes, both in cells that naturally lack them, and those that have had them experimentally removed. The results of these studies suggest that an appreciation of microtubule network organization- both before and after nuclear envelope breakdown (NEB) - is the key to understanding the mechanisms that regulate spindle assembly and the generation of bipolarity.

SummaryThe 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 [1–3]. However, vertebrate somatic cells normally assemble a connected pair of polarized, astral MT arrays—termed an amphiaster (“a star on both sides” [4])—that is formed by the splitting and separation of the microtubule-organizing center (MTOC) well before nuclear envelope breakdown (NEB) [5]. Whether amphiaster formation requires splitting of duplicated centrosomes is not known. We found that when centrosomes were removed from living vertebrate cells early in their cell cycle, an acentriolar MTOC reassembled, and, prior to NEB, a functional amphiastral spindle formed. Cytoplasmic dynein, dynactin, and pericentrin are all recruited to the interphase aMTOC, and the activity of kinesin-5 is needed for amphiaster formation. Mitosis proceeded on time and these karyoplasts divided in two. However, ∼35% of aMTOCs failed to split and separate before NEB, and these entered mitosis with persistent monastral spindles. Chromatin-associated RAN-GTP—the small GTPase Ran in its GTP bound state—could not restore bipolarity to monastral spindles, and these cells exited mitosis as single daughters. Our data reveal the novel finding that MTOC separation and amphiaster formation does not absolutely require the centrosome, but, in its absence, the fidelity of bipolar spindle assembly is highly compromised.

The synthetic Vitamin A analog fenretinide is a promising chemotherapeutic agent. In the current paper, the role of PKC was examined in fenretinide-induced apoptosis in lymphoid leukemia cells. Levels of proapoptotic cleaved PKC positively correlated with drug sensitivity. Fenretinide promoted reactive oxygen species (ROS) generation. The antioxidant Vitamin C prevented fenretinide-induced PKC cleavage and protected cells from fenretinide. Suppression of PKC expression by shRNA sensitized cells to fenretinide-induced apoptosis possibly by a mechanism involving ROS production. A previous study demonstrated that fenretinide promotes degradation of antiapoptotic MCL-1 in ALL cells via JNK. Now we have found that fenretinide-induced MCL-1 degradation may involve PKC as cleavage of the kinase correlated with loss of MCL-1 even in cells when JNK was not activated. These results suggest that PKC may play a complex role in fenretinide-induced apoptosis and may be targeted in antileukemia strategies that utilize fenretinide.

In mammalian cultured cells the initiation of cytokinesis is regulated – both temporally and spatially – by the overlapping, anti-parallel microtubules of the spindle midzone. This region recruits several key central spindle components: PRC-1, polo-like kinase 1 (Plk-1), the centralspindlin complex, and the chromosome passenger complex (CPC), which together serve to stabilize the microtubule overlap, and also to coordinate the assembly of the cortical actin/myosin cytoskeleton necessary to physically cleave the cell in two. The localization of these crucial elements to the spindle midzone requires members of the kinesin superfamily of microtubule-based motor proteins. Here we focus on reviewing the role played by a variety of kinesins in both building and operating the spindle midzone machinery during cytokinesis.

During anaphase, the nonkinetochore microtubules in the spindle midzone become compacted into the central spindle, a structure which is required to both initiate and complete cytokinesis. We show that Tektin 2 (Tek2) associates with the spindle poles throughout mitosis, organizes the spindle midzone microtubules during anaphase, and assembles into the midbody matrix surrounding the compacted midzone microtubules during cytokinesis. Tek2 small interfering RNA (siRNA) disrupts central spindle organization and proper localization of MKLP1, PRC1, and Aurora B to the midzone and prevents the formation of a midbody matrix. Video microscopy revealed that loss of Tek2 results in binucleate cell formation by aberrant fusion of daughter cells after cytokinesis. Although a myosin II inhibitor, blebbistatin, prevents actin-myosin contractility, the microtubules of the central spindle are compacted. Strikingly, Tek2 siRNA abolishes this actin-myosin–independent midzone microtubule compaction. Thus, Tek2-dependent organization of the central spindle during anaphase is essential for proper midbody formation and the segregation of daughter cells after cytokinesis.

Cytoplasmic dynein functions at several sites during mitosis; however, the basis of targeting to each site remains unclear. Tandem mass spectrometry analysis of mitotic dynein revealed a phosphorylation site in the dynein intermediate chains (ICs) that mediates binding to kinetochores. IC phosphorylation directs binding to zw10 rather than dynactin, and this interaction is needed for kinetochore dynein localization. Phosphodynein associates with kinetochores from nuclear envelope breakdown to metaphase, but bioriented microtubule (MT) attachment and chromosome alignment induce IC dephosphorylation. IC dephosphorylation stimulates binding to dynactin and poleward streaming. MT depolymerization, release of kinetochore tension, and a PP1-γ mutant each inhibited IC dephosphorylation, leading to the retention of phosphodynein at kinetochores and reduced poleward streaming. The depletion of kinetochore dynactin by moderate levels of p50(dynamitin) expression disrupted the ability of dynein to remove checkpoint proteins by streaming at metaphase but not other aspects of kinetochore dynein activity. Together, these results suggest a new model for localization of kinetochore dynein and the contribution of kinetochore dynactin.

The successful division of the cell depends on several morphological events that must be coordinated in a cell cycle dependent fashion. Crucial to this process is the remodeling of the cytoskeleton to both establish the bipolar spindle during mitosis, and cleave the cell into two daughters during cytokinesis. From the standpoint of the cytoskeleton, this process begins during interphase with the duplication of the centrosome; it is the two daughter centrosomes that will assemble the poles of the mitotic spindle and establish its necessary bipolarity. Following the cell cycle transition into mitosis, the spindle must assemble in order to properly align the sister chromatids at the center of the cell, and release the “wait anaphase checkpoint”. As the spindle transports the disjoined sister chromatids to the spindle poles, the cell must rapidly undergo cytokinesis, cleaving the cell into two – the plane of cleavage being established by the spindle itself. Understanding the temporal regulation and molecular basis for these events has come from extensive experiments using a variety of model systems, and has benefited from cell biological, molecular genetic, and biophysical approaches. One of the earliest and most important model systems for studying mitosis is the sea urchin zygote. With their large size, rapid/synchronous cell cycles, and advantages for conduct of biochemical and cytological investigations on the same system, fertilized sea urchin eggs have revealed many of the fundamental properties of centrosome duplication, cell division and cytokinesis. Here we review several key studies that have utilized the sea urchin zygote to explore mechanisms that coordinate and drive two of the major cytoskeletal events of mitotic division – centrosome duplication and cytokinesis.

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. Here we identify a conserved feedback mechanism that monitors missegregating chromosomes during anaphase through the differential phosphorylation of histone H3.3 at Ser31. We do this by inducing transient chromosome missegregation in diploid cells. During anaphase, H3.3 Ser31 is phosphorylated along the arms of lagging or misaligned chromosomes. Within minutes, Ser31 phosphorylation (Ser31P) spreads to all of the chromatids of both daughter cells, which persists into G1. Masking H3.3 Ser31P by antibody microinjection prevents nuclear p53 accumulation in the aneuploid daughters. Previous work demonstrated that prolonged prometaphase and DNA damage during abnormal mitosis can activate p53. We show that p53 activation in response to chromosome missegregation can occur without prolonged mitosis or DNA damage. Our study provides insight into how aneuploidy caused by chromosome missegregation is normally monitored and suppressed.

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. Because the centrosome lies at the center of the array of microtubule minus-ends, and the essential one-to-two duplication of the centrosome prior to mitosis is linked to cell cycle progression, this organelle has long been implicated as a device to generate spindle bipolarity. However, this classic model for spindle assembly is challenged by observations and experimental manipulations demonstrating that acentrosomal cells can and do form bipolar spindles, both mitotic and meiotic. Indeed, recent comprehensive proteomic analysis of centrosome-dependent versus independent mitotic spindle assembly mechanisms reveals a large, common set of genes required for both processes, with very few genes needed to differentiate between the two. While these studies cast doubt on an absolute role for the centrosome in establishing spindle polarity, it is clear that having too few or too many centrosomes results in abnormal chromosome segregation and aneuploidy. Here we review the case both for and against the role of the centrioles and centrosomes in ensuring proper assembly of a bipolar spindle, an essential element in the maintenance of genomic stability.

Ceramide 1-Phosphate (C1P) is a biologically active sphingolipid that stimulates cell proliferation, macrophage migration, and inflammatory events. We recently characterized a novel human lipid transfer protein encoded by GLTPD1 on chromosome 1 (locus 1p36.33). We named the protein, ceramide 1-phosphate transfer protein (CPTP) due to its lipid specificity and discovered that CPTP transports C1P to the plasma membrane from TGN after synthesis by ceramide kinase. CPTP depletion by RNAi leads to C1P accumulation in TGN (~6-fold), stimulating cPLA2α and inducing pro-inflammatory eicosanoid production (Simanshu et al., Nature 500: 463-67, 2013). Here, we investigated whether CPTP depletion leads to programmed cell death and discovered an autophagic response in human cervical cancer cell lines (HeLa) and human lung adenocarcinoma epithelial cells (A549). Transfection with ShRNA (pSuper.GFP.ShRNA.CPTP vector) resulted in GFP-LC3 puncta and acidophilic acridine orange staining by epifluorescence microscopy and be...

Centrosomes were microsurgically removed from BSC-1 African green monkey kidney cells before the completion of S phase. Karyoplasts (acentrosomal cells) entered and completed mitosis. However, postmitotic karyoplasts arrested before S phase, whereas adjacent control cells divided repeatedly. Postmitotic karyoplasts assembled a microtubule-organizing center containing γ-tubulin and pericentrin, but did not regenerate centrioles. These observations reveal the existence of an activity associated with core centrosomal structures—distinct from elements of the microtubule-organizing center—that is required for the somatic cell cycle to progress through G 1 into S phase. Once the cell is in S phase, these core structures are not needed for the G 2 -M phase transition.

Rac1 is a small G-protein in the Ras superfamily that has been implicated in the control of cell growth, adhesion, and the actin-based cytoskeleton. To investigate the role of Rac1 during motile processes, we have established Dictyostelium cell lines that conditionally overexpress epitope-tagged Dictyostelium discoideum wild-type Rac1B (DdRac1B) or a mutant DdRac1B protein. Expression of endogenous levels of myc- or GFP-tagged wild-type DdRac1B had minimal effect on cellular morphologies and behaviors. By contrast, expression of a constitutively active mutant (G12-->V or Q61-->L) or a dominant negative mutant (T17-->N) generated amoebae with characteristic cellular defects. The morphological appearance of actin-containing structures, intracellular levels of F-actin, and cellular responses to chemoattractant closely paralleled the amount of active DdRac1B, indicating a role in upregulating actin cytoskeletal activities. Expression of any of the three mutants inhibited cell growth and cytokinesis, and delayed multicellular development, suggesting that DdRac1B plays important regulatory role(s) during these processes. No significant effects were observed on binding or internalization of latex beads in suspension or on intracellular membrane trafficking. Cells expressing DdRac1B-G12V exhibited defects in fluid-phase endocytosis and the longest developmental delays; DdRac1B-Q61L produced the strongest cytokinesis defect; and DdRac1B-T17N generated intermediate phenotypes. These conditionally expressed DdRac1B proteins should facilitate the identification and characterization of the Rac1 signaling pathway in an organism that is amenable to both biochemical and molecular genetic manipulations.

The abnormally high number of centrosomes found in many human tumor cells can lead directly to aneuploidy and genomic instability through the formation of multipolar mitotic spindles. To facilitate investigation of the mechanisms that control centrosome reproduction, a frog egg extract arrested in S phase of the cell cycle that supported repeated assembly of daughter centrosomes was developed. Multiple rounds of centrosome reproduction were blocked by selective inactivation of cyclin-dependent kinase 2–cyclin E (Cdk2-E) and were restored by addition of purified Cdk2-E. Confocal immunomicroscopy revealed that cyclin E was localized at the centrosome. These results demonstrate that Cdk2-E activity is required for centrosome duplication during S phase and suggest a mechanism that could coordinate centrosome reproduction with cycles of DNA synthesis and mitosis.

Centrosomes repeatedly reproduce in sea urchin zygotes arrested in S phase, whether cyclin-dependent kinase 1–cyclin B (Cdk1-B) activity remains at prefertilization levels or rises to mitotic values. In contrast, when zygotes are arrested in mitosis using cyclin B Δ-90, anaphase occurs at the normal time, yet centrosomes do not reproduce. Together, these results reveal the cell cycle stage specificity for centrosome reproduction and demonstrate that neither the level nor the cycling of Cdk1-B activity coordinate centrosome reproduction with nuclear events. In addition, the proteolytic events of the metaphase–anaphase transition do not control when centrosomes duplicate. When we block protein synthesis at first prophase, the zygotes divide and arrest before second S phase. Both blastomeres contain just two complete centrosomes, which indicates that the cytoplasmic conditions between mitosis and S phase support centrosome reproduction. However, the fact that these daughter centrosomes do not reproduce again under such supportive conditions suggests that they are lacking a component required for reproduction. The repeated reproduction of centrosomes during S phase arrest points to the existence of a necessary “licensing” event that restores this component to daughter centrosomes during S phase, preparing them to reproduce in the next cell cycle.

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. Because the centrosome lies at the center of the array of microtubule minus-ends, and the essential one-to-two duplication of the centrosome prior to mitosis is linked to cell cycle progression, this organelle has long been implicated as a device to generate spindle bipolarity. However, this classic model for spindle assembly is challenged by observations and experimental manipulations demonstrating that acentrosomal cells can and do form bipolar spindles, both mitotic and meiotic. Indeed, recent comprehensive proteomic analysis of centrosome-dependent versus independent mitotic spindle assembly mechanisms reveals a large, common set of genes required for both processes, with very few genes needed to differentiate between the two. While these studies cast doubt on an absolute role for the centrosome in establishing spindle polarity, it is clear that having too few or too many centrosomes results in abnormal chromosome segregation and aneuploidy. Here we review the case both for and against the role of the centrioles and centrosomes in ensuring proper assembly of a bipolar spindle, an essential element in the maintenance of genomic stability.

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.

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. In this chapter, we discuss image file formats, and the various components of these files, such as bit-depth, sampling rate, color theory, and compression, from the perspective of the non-computer scientist. We also discuss the problem of proprietary file formats, and how these often are incompatible with certain types of imaging software. We present several solutions to this issue. Finally, we present the use of digital movie formats, compression routines, and provide some real world examples for optimizing the generation of digital movies.

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. Here we discuss some of the basic aspects of digital data storage and use, including file types, storage media, and the choice between commercial and open-source software. Often, open-source software is written by a user or group of users, and then distributed to the scientific community at large. These can be important tools, but there are some hidden costs to this freeware that we will discuss. We will also compare open-source software to commercial imaging software, which is often written for use by non-computer scientists.

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.

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. In this chapter, we discuss image file formats, and the various components of these files, such as bit-depth, sampling rate, color theory, and compression, from the perspective of the non-computer scientist. We also discuss the problem of proprietary file formats, and how these often are incompatible with certain types of imaging software. We present several solutions to this issue. Finally, we present the use of digital movie formats, compression routines, and provide some real world examples for optimizing the generation of digital movies.