Somatic polyploidy supports biosynthesis and tissue function by increasing transcriptional output.

Cell size and biosynthetic capacity generally increase with increased DNA content. Polyploidy has therefore been proposed to be an adaptive strategy to increase cell size in specialized tissues with high biosynthetic demands. However, if and how DNA concentration limits cellular biosynthesis in vivo is not well understood, and the impacts of polyploidy in non-disease states is not well studied. Here, we show that polyploidy in the C. elegans intestine is critical for cell growth and yolk biosynthesis, a central role of this organ. Artificially lowering the DNA/cytoplasm ratio by reducing polyploidization in the intestine gave rise to smaller cells with more dilute mRNA. Highly-expressed transcripts were more sensitive to this mRNA dilution, whereas lowly-expressed genes were partially compensated - in part by loading more RNA Polymerase II on the remaining genomes. DNA-dilute cells had normal total protein concentration, which we propose is achieved by increasing production of translational machinery at the expense of specialized, cell-type specific proteins.

Context matters: Lessons in epithelial polarity from the Caenorhabditis elegans intestine and other tissues.

Epithelia are tissues with diverse morphologies and functions across metazoans, ranging from vast cell sheets encasing internal organs to internal tubes facilitating nutrient uptake, all of which require establishment of apical-basolateral polarity axes. While all epithelia tend to polarize the same components, how these components are deployed to drive polarization is largely context-dependent and likely shaped by tissue-specific differences in development and ultimate functions of polarizing primordia. The nematode Caenorhabditis elegans (C. elegans) offers exceptional imaging and genetic tools and possesses unique epithelia with well-described origins and roles, making it an excellent model to investigate polarity mechanisms. In this review, we highlight the interplay between epithelial polarization, development, and function by describing symmetry breaking and polarity establishment in a particularly well-characterized epithelium, the C. elegans intestine. We compare intestinal polarization to polarity programs in two other C. elegans epithelia, the pharynx and epidermis, correlating divergent mechanisms with tissue-specific differences in geometry, embryonic environment, and function. Together, we emphasize the importance of investigating polarization mechanisms against the backdrop of tissue-specific contexts, while also underscoring the benefits of cross-tissue comparisons of polarity.

Cryo-EM structure of the human Sirtuin 6-nucleosome complex.

Sirtuin 6 (SIRT6) is a multifaceted protein deacetylase/deacylase and a major target for small-molecule modulators of longevity and cancer. In the context of chromatin, SIRT6 removes acetyl groups from histone H3 in nucleosomes, but the molecular basis for its nucleosomal substrate preference is unknown. Our cryo-electron microscopy structure of human SIRT6 in complex with the nucleosome shows that the catalytic domain of SIRT6 pries DNA from the nucleosomal entry-exit site and exposes the histone H3 N-terminal helix, while the SIRT6 zinc-binding domain binds to the histone acidic patch using an arginine anchor. In addition, SIRT6 forms an inhibitory interaction with the C-terminal tail of histone H2A. The structure provides insights into how SIRT6 can deacetylate both H3 K9 and H3 K56.

Cryo-EM structure of the human Sirtuin 6-nucleosome complex.

Sirtuin 6 (SIRT6) is a multifaceted protein deacetylase/deacylase and a major target for small-molecule modulators of longevity and cancer. In the context of chromatin, SIRT6 removes acetyl groups from histone H3 in nucleosomes, but the molecular basis for its nucleosomal substrate preference is unknown. Our cryo-electron microscopy structure of human SIRT6 in complex with the nucleosome shows that the catalytic domain of SIRT6 pries DNA from the nucleosomal entry-exit site and exposes the histone H3 N-terminal helix, while the SIRT6 zinc-binding domain binds to the histone acidic patch using an arginine anchor. In addition, SIRT6 forms an inhibitory interaction with the C-terminal tail of histone H2A. The structure provides insights into how SIRT6 can deacetylate both H3 K9 and H3 K56. Teaser: The structure of the SIRT6 deacetylase/nucleosome complex suggests how the enzyme acts on both histone H3 K9 and K56 residues.

Separable mechanisms drive local and global polarity establishment in the Caenorhabditiselegans intestinal epithelium.

Apico-basolateral polarization is essential for epithelial cells to function as selective barriers and transporters, and to provide mechanical resilience to organs. Epithelial polarity is established locally, within individual cells to establish distinct apical, junctional and basolateral domains, and globally, within a tissue where cells coordinately orient their apico-basolateral axes. Using live imaging of endogenously tagged proteins and tissue-specific protein depletion in the Caenorhabditiselegans embryonic intestine, we found that local and global polarity establishment are temporally and genetically separable. Local polarity is initiated prior to global polarity and is robust to perturbation. PAR-3 is required for global polarization across the intestine but local polarity can arise in its absence, as small groups of cells eventually established polarized domains in PAR-3-depleted intestines in a HMR-1 (E-cadherin)-dependent manner. Despite the role of PAR-3 in localizing PKC-3 to the apical surface, we additionally found that PAR-3 and PKC-3/aPKC have distinct roles in the establishment and maintenance of local and global polarity. Taken together, our results indicate that different mechanisms are required for local and global polarity establishment in vivo.

Won't You be My Neighbor: How Epithelial Cells Connect Together to Build Global Tissue Polarity.

Epithelial tissues form continuous barriers to protect against external environments. Within these tissues, epithelial cells build environment-facing apical membranes, junction complexes that anchor neighbors together, and basolateral surfaces that face other cells. Critically, to form a continuous apical barrier, neighboring epithelial cells must align their apico-basolateral axes to create global polarity along the entire tissue. Here, we will review mechanisms of global tissue-level polarity establishment, with a focus on how neighboring epithelial cells of different origins align their apical surfaces. Epithelial cells with different developmental origins and/or that polarize at different times and places must align their respective apico-basolateral axes. Connecting different epithelial tissues into continuous sheets or tubes, termed epithelial fusion, has been most extensively studied in cases where neighboring cells initially dock at an apical-to-apical interface. However, epithelial cells can also meet basal-to-basal, posing several challenges for apical continuity. Pre-existing basement membrane between the tissues must be remodeled and/or removed, the cells involved in docking are specialized, and new cell-cell adhesions are formed. Each of these challenges can involve changes to apico-basolateral polarity of epithelial cells. This minireview highlights several in vivo examples of basal docking and how apico-basolateral polarity changes during epithelial fusion. Understanding the specific molecular mechanisms of basal docking is an area ripe for further exploration that will shed light on complex morphogenetic events that sculpt developing organisms and on the cellular mechanisms that can go awry during diseases involving the formation of cysts, fistulas, atresias, and metastases.

Apical PAR complex proteins protect against programmed epithelial assaults to create a continuous and functional intestinal lumen.

Sustained polarity and adhesion of epithelial cells is essential for the protection of our organs and bodies, and this epithelial integrity emerges during organ development amidst numerous programmed morphogenetic assaults. Using the developing Caenorhabditis elegans intestine as an in vivo model, we investigated how epithelia maintain their integrity through cell division and elongation to build a functional tube. Live imaging revealed that apical PAR complex proteins PAR-6/Par6 and PKC-3/aPkc remained apical during mitosis while apical microtubules and microtubule-organizing center (MTOC) proteins were transiently removed. Intestine-specific depletion of PAR-6, PKC-3, and the aPkc regulator CDC-42/Cdc42 caused persistent gaps in the apical MTOC as well as in other apical and junctional proteins after cell division and in non-dividing cells that elongated. Upon hatching, gaps coincided with luminal constrictions that blocked food, and larvae arrested and died. Thus, the apical PAR complex maintains apical and junctional continuity to construct a functional intestinal tube.

Microtubule organization across cell types and states.

Encircling and traversing the cell are architectural struts and dynamic intracellular highways made of cylindrical polymers called microtubules. Built from structurally asymmetric subunits of αß-tubulin heterodimers, microtubules have an inherent structural polarity with a slow-growing minus end and a comparatively dynamic plus end that grows and shrinks. Thus, a key feature of microtubules is that each polymer is polarized, allowing for the execution of cellular tasks that are directional in nature. For example, microtubules build polarized highways allowing directional intracellular transport, generate directional force such as in chromosome alignment and segregation, provide structural support for cell shape, and assemble into highly ordered polar structures like centrioles and cilia. The output of these microtubule-based functions is the performance of different tasks, including establishment and maintenance of cellular polarity, secretion and absorption, cell-cell communication, migration, mechanical resiliency, and mitosis. Different cells accomplish these functions by using distinct sites within the cell called microtubule-organizing centers (MTOCs) to build cell-specific microtubule arrangements. While the specific requirement for microtubules in many in vivo cell types is unknown, disrupting even a subset of microtubule-supported functions is often lethal and is associated with many diseases (e.g., cancer and neuropathies), suggesting that specific patterns of microtubule organization are likely important for cellular function in vivo. This Primer focuses on how differentiated animal and plant cells use distinct MTOCs to generate specific microtubule arrangements, how those arrangements support cellular functions, and how cells rearrange their microtubules to accommodate changing cellular tasks.

A Polarizing Issue: Diversity in the Mechanisms Underlying Apico-Basolateral Polarization In Vivo.

Polarization along an apico-basolateral axis is a hallmark of epithelial cells and is essential for their selective barrier and transporter functions, as well as for their ability to provide mechanical resiliency to organs. Loss of polarity along this axis perturbs development and is associated with a wide number of diseases. We describe three steps involved in polarization: symmetry breaking, polarity establishment, and polarity maintenance. While the proteins involved in these processes are highly conserved among epithelial tissues and species, the execution of these steps varies widely and is context dependent. We review both theoretical principles underlying these steps and recent work demonstrating how apico-basolateral polarity is established in vivo in different tissues, highlighting how developmental and physiological contexts play major roles in the execution of the epithelial polarity program.

Yeast Sirtuin Family Members Maintain Transcription Homeostasis to Ensure Genome Stability.

The mammalian sirtuin, SIRT6, is a key tumor suppressor that maintains genome stability and regulates transcription, though how SIRT6 family members control genome stability is unclear. Here, we use multiple genome-wide approaches to demonstrate that the yeast SIRT6 homologs, Hst3 and Hst4, prevent genome instability by tuning levels of both coding and noncoding transcription. While nascent RNAs are elevated in the absence of Hst3 and Hst4, a global impact on steady-state mRNAs is masked by the nuclear exosome, indicating that sirtuins and the exosome provide two levels of regulation to maintain transcription homeostasis. We find that, in the absence of Hst3 and Hst4, increased transcription is associated with excessive DNA-RNA hybrids (R-loops) that appear to lead to new DNA double-strand breaks. Importantly, dissolution of R-loops suppresses the genome instability phenotypes of hst3 hst4 mutants, suggesting that the sirtuins maintain genome stability by acting as a rheostat to prevent promiscuous transcription.

A two-step mechanism for the inactivation of microtubule organizing center function at the centrosome.

The centrosome acts as a microtubule organizing center (MTOC), orchestrating microtubules into the mitotic spindle through its pericentriolar material (PCM). This activity is biphasic, cycling through assembly and disassembly during the cell cycle. Although hyperactive centrosomal MTOC activity is a hallmark of some cancers, little is known about how the centrosome is inactivated as an MTOC. Analysis of endogenous PCM proteins in C. elegans revealed that the PCM is composed of partially overlapping territories organized into an inner and outer sphere that are removed from the centrosome at different rates and using different behaviors. We found that phosphatases oppose the addition of PCM by mitotic kinases, ultimately catalyzing the dissolution of inner sphere PCM proteins at the end of mitosis. The nature of the PCM appears to change such that the remaining aging PCM outer sphere is mechanically ruptured by cortical pulling forces, ultimately inactivating MTOC function at the centrosome.

Tissue-specific degradation of essential centrosome components reveals distinct microtubule populations at microtubule organizing centers.

Non-centrosomal microtubule organizing centers (ncMTOCs) are found in most differentiated cells, but how these structures regulate microtubule organization and dynamics is largely unknown. We optimized a tissue-specific degradation system to test the role of the essential centrosomal microtubule nucleators γ-tubulin ring complex (γ-TuRC) and AIR-1/Aurora A at the apical ncMTOC, where they both localize in Caenorhabditis elegans embryonic intestinal epithelial cells. As at the centrosome, the core γ-TuRC component GIP-1/GCP3 is required to recruit other γ-TuRC components to the apical ncMTOC, including MZT-1/MZT1, characterized here for the first time in animal development. In contrast, AIR-1 and MZT-1 were specifically required to recruit γ-TuRC to the centrosome, but not to centrioles or to the apical ncMTOC. Surprisingly, microtubules remain robustly organized at the apical ncMTOC upon γ-TuRC and AIR-1 co-depletion, and upon depletion of other known microtubule regulators, including TPXL-1/TPX2, ZYG-9/ch-TOG, PTRN-1/CAMSAP, and NOCA-1/Ninein. However, loss of GIP-1 removed a subset of dynamic EBP-2/EB1-marked microtubules, and the remaining dynamic microtubules grew faster. Together, these results suggest that different microtubule organizing centers (MTOCs) use discrete proteins for their function, and that the apical ncMTOC is composed of distinct populations of γ-TuRC-dependent and -independent microtubules that compete for a limited pool of resources.

Identification of and Molecular Basis for SIRT6 Loss-of-Function Point Mutations in Cancer.

Chromatin factors have emerged as the most frequently dysregulated family of proteins in cancer. We have previously identified the histone deacetylase SIRT6 as a key tumor suppressor, yet whether point mutations are selected for in cancer remains unclear. In this manuscript, we characterized naturally occurring patient-derived SIRT6 mutations. Strikingly, all the mutations significantly affected either stability or catalytic activity of SIRT6, indicating that these mutations were selected for in these tumors. Further, the mutant proteins failed to rescue sirt6 knockout (SIRT6 KO) cells, as measured by the levels of histone acetylation at glycolytic genes and their inability to rescue the tumorigenic potential of these cells. Notably, the main activity affected in the mutants was histone deacetylation rather than demyristoylation, pointing to the former as the main tumor-suppressive function for SIRT6. Our results identified cancer-associated point mutations in SIRT6, cementing its function as a tumor suppressor in human cancer.

SPD-2/CEP192 and CDK Are Limiting for Microtubule-Organizing Center Function at the Centrosome.

The centrosome acts as the microtubule-organizing center (MTOC) during mitosis in animal cells. Microtubules are nucleated and anchored by γ-tubulin ring complexes (γ-TuRCs) embedded within the centrosome's pericentriolar material (PCM). The PCM is required for the localization of γ-TuRCs, and both are steadily recruited to the centrosome, culminating in a peak in MTOC function in metaphase. In differentiated cells, the centrosome is often attenuated as an MTOC and MTOC function is reassigned to non-centrosomal sites such as the apical membrane in epithelial cells, the nuclear envelope in skeletal muscle, and down the lengths of axons and dendrites in neurons. Hyperactive MTOC function at the centrosome is associated with epithelial cancers and with invasive behavior in tumor cells. Little is known about the mechanisms that limit MTOC activation at the centrosome. Here, we find that MTOC function at the centrosome is completely inactivated during cell differentiation in C. elegans embryonic intestinal cells and MTOC function is reassigned to the apical membrane. In cells that divide after differentiation, the cellular MTOC state switches between the membrane and the centrosome. Using cell fusion experiments in live embryos, we find that the centrosome MTOC state is dominant and that the inactive MTOC state of the centrosome is malleable; fusion of a mitotic cell to a differentiated or interphase cell results in rapid reactivation of the centrosome MTOC. We show that conversion of MTOC state involves the conserved centrosome protein SPD-2/CEP192 and CDK activity from the mitotic cell.

Metabolic regulation of histone post-translational modifications.

Histone post-translational modifications regulate transcription and other DNA-templated functions. This process is dynamically regulated by specific modifying enzymes whose activities require metabolites that either serve as cosubstrates or act as activators/inhibitors. Therefore, metabolism can influence histone modification by changing local concentrations of key metabolites. Physiologically, the epigenetic response to metabolism is important for nutrient sensing and environment adaption. In pathologic states, the connection between metabolism and histone modification mediates epigenetic abnormality in complex disease. In this review, we summarize recent studies of the molecular mechanisms involved in metabolic regulation of histone modifications and discuss their biological significance.

Quantitative analysis and modeling of katanin function in flagellar length control.

Flagellar length control in Chlamydomonas reinhardtii provides a simple model system in which to investigate the general question of how cells regulate organelle size. Previous work demonstrated that Chlamydomonas cytoplasm contains a pool of flagellar precursor proteins sufficient to assemble a half-length flagellum and that assembly of full-length flagella requires synthesis of additional precursors to augment the preexisting pool. The regulatory systems that control the synthesis and regeneration of this pool are not known, although transcriptional regulation clearly plays a role. We used quantitative analysis of length distributions to identify candidate genes controlling pool regeneration and found that a mutation in the p80 regulatory subunit of katanin, encoded by the PF15 gene in Chlamydomonas, alters flagellar length by changing the kinetics of precursor pool utilization. This finding suggests a model in which flagella compete with cytoplasmic microtubules for a fixed pool of tubulin, with katanin-mediated severing allowing easier access to this pool during flagellar assembly. We tested this model using a stochastic simulation that confirms that cytoplasmic microtubules can compete with flagella for a limited tubulin pool, showing that alteration of cytoplasmic microtubule severing could be sufficient to explain the effect of the pf15 mutations on flagellar length.

Cell interactions and patterned intercalations shape and link epithelial tubes in C. elegans.

Many animal organs are composed largely or entirely of polarized epithelial tubes, and the formation of complex organ systems, such as the digestive or vascular systems, requires that separate tubes link with a common polarity. The Caenorhabditis elegans digestive tract consists primarily of three interconnected tubes-the pharynx, valve, and intestine-and provides a simple model for understanding the cellular and molecular mechanisms used to form and connect epithelial tubes. Here, we use live imaging and 3D reconstructions of developing cells to examine tube formation. The three tubes develop from a pharynx/valve primordium and a separate intestine primordium. Cells in the pharynx/valve primordium polarize and become wedge-shaped, transforming the primordium into a cylindrical cyst centered on the future lumenal axis. For continuity of the digestive tract, valve cells must have the same, radial axis of apicobasal polarity as adjacent intestinal cells. We show that intestinal cells contribute to valve cell polarity by restricting the distribution of a polarizing cue, laminin. After developing apicobasal polarity, many pharyngeal and valve cells appear to explore their neighborhoods through lateral, actin-rich lamellipodia. For a subset of cells, these lamellipodia precede more extensive intercalations that create the valve. Formation of the valve tube begins when two valve cells become embedded at the left-right boundary of the intestinal primordium. Other valve cells organize symmetrically around these two cells, and wrap partially or completely around the orthogonal, lumenal axis, thus extruding a small valve tube from the larger cyst. We show that the transcription factors DIE-1 and EGL-43/EVI1 regulate cell intercalations and cell fates during valve formation, and that the Notch pathway is required to establish the proper boundary between the pharyngeal and valve tubes.

Nitration of Hsp90 induces cell death.

Oxidative stress is a widely recognized cause of cell death associated with neurodegeneration, inflammation, and aging. Tyrosine nitration in these conditions has been reported extensively, but whether tyrosine nitration is a marker or plays a role in the cell-death processes was unknown. Here, we show that nitration of a single tyrosine residue on a small proportion of 90-kDa heat-shock protein (Hsp90), is sufficient to induce motor neuron death by the P2X7 receptor-dependent activation of the Fas pathway. Nitrotyrosine at position 33 or 56 stimulates a toxic gain of function that turns Hsp90 into a toxic protein. Using an antibody that recognizes the nitrated Hsp90, we found immunoreactivity in motor neurons of patients with amyotrophic lateral sclerosis, in an animal model of amyotrophic lateral sclerosis, and after experimental spinal cord injury. Our findings reveal that cell death can be triggered by nitration of a single protein and highlight nitrated Hsp90 as a potential target for the development of effective therapies for a large number of pathologies.

A role for the centrosome and PAR-3 in the hand-off of MTOC function during epithelial polarization.

BACKGROUND: The centrosome is the major microtubule organizing center (MTOC) in dividing cells and in many postmitotic, differentiated cells. In other cell types, however, MTOC function is reassigned from the centrosome to noncentrosomal sites. Here, we analyze how MTOC function is reassigned to the apical membrane of C. elegans intestinal cells. RESULTS: After the terminal intestinal cell division, the centrosomes and nuclei move near the future apical membranes, and the postmitotic centrosomes lose all, or nearly all, of their associated microtubules. We show that microtubule-nucleating proteins such as γ-tubulin and CeGrip-1 that are centrosome components in dividing cells become localized to the apical membrane, which becomes highly enriched in microtubules. Our results suggest that centrosomes are critical to specify the apical membrane as the new MTOC. First, γ-tubulin appears to redistribute directly from the migrating centrosome onto the lateral then apical membrane. Second, γ-tubulin fails to accumulate apically in wild-type cells following laser ablation of the centrosome. We show that centrosomes localize apically by first moving toward lateral foci of the conserved polarity proteins PAR-3 and PAR-6 and then move together with these foci toward the future apical surface. Embryos lacking PAR-3 fail to localize their centrosomes apically and have aberrant localization of γ-tubulin and CeGrip-1. CONCLUSIONS: These data suggest that PAR proteins contribute to apical polarity in part by determining centrosome position and that the reassignment of MTOC function from centrosomes to the apical membrane is associated with a physical hand-off of nucleators of microtubule assembly.

Structure and biochemical functions of SIRT6.

SIRT6 is a member of the evolutionarily conserved sirtuin family of NAD(+)-dependent protein deacetylases and functions in genomic stability and transcriptional control of glucose metabolism. Early reports suggested that SIRT6 performs ADP-ribosylation, whereas more recent studies have suggested that SIRT6 functions mainly as a histone deacetylase. Thus, the molecular functions of SIRT6 remain uncertain. Here, we perform biochemical, kinetic, and structural studies to provide new mechanistic insight into the functions of SIRT6. Utilizing three different assays, we provide biochemical and kinetic evidence that SIRT6-dependent histone deacetylation produces O-acetyl-ADP-ribose but at a rate ∼1,000 times slower than other highly active sirtuins. To understand the molecular basis for such low deacetylase activity, we solved the first crystal structures of this class IV sirtuin in complex with ADP-ribose and the non-hydrolyzable analog of O-acetyl-ADP-ribose, 2'-N-acetyl-ADP-ribose. The structures revealed unique features of human SIRT6, including a splayed zinc-binding domain and the absence of a helix bundle that in other sirtuin structures connects the zinc-binding motif and Rossmann fold domain. SIRT6 also lacks the conserved, highly flexible, NAD(+)-binding loop and instead contains a stable single helix. These differences led us to hypothesize that SIRT6, unlike all other studied sirtuins, would be able to bind NAD(+) in the absence of an acetylated substrate. Indeed, we found that SIRT6 binds NAD(+) with relatively high affinity (K(d) = 27 ± 1 µM) in the absence of an acetylated substrate. Isothermal titration calorimetry and tryptophan fluorescence binding assays suggested that ADP-ribose and NAD(+) induce different structural perturbations and that NADH does not bind to SIRT6. Collectively, these new insights imply a unique activating mechanism and/or the possibility that SIRT6 could act as an NAD(+) metabolite sensor.