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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.

SummaryMicrotubule doublets (MTDs) are a well conserved compound microtubule structure found primarily in cilia. However, the mechanisms by which MTDs form and are maintainedin vivoremain poorly understood. Here, we characterize microtubule-associated protein 9 (MAP9) as a novel MTD-associated protein. We demonstrate thatC. elegansMAPH-9, a MAP9 homolog, is present during MTD assembly and localizes exclusively to MTDs, a preference that is in part mediated by tubulin polyglutamylation. Loss of MAPH-9 caused ultrastructural MTD defects, dysregulated axonemal motor velocity, and perturbed cilia function. As we found that the mammalian ortholog MAP9 localized to axonemes in cultured mammalian cells and mouse tissues, we propose that MAP9/MAPH-9 plays a conserved role in supporting the structure of axonemal MTDs and regulating ciliary motors.

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.

SummaryTissue-wide patterning is essential to multicellular development, requiring cells to individually generate polarity axes and coordinate them in space and time with neighbors. Using the C. elegans intestinal epithelium, we identified a patterning mechanism informed by stabilized cell/cell contact and executed via the scaffolding protein PAR-3 and the transmembrane protein E-cadherin/HMR-1. Intestinal cells break symmetry as PAR-3 and HMR-1 recruit apical determinants into micron-scale ‘local polarity complexes’ (LPCs) at homotypic contacts. LPCs undergo a HMR-1-based migration to a common tissue midline, thereby establishing tissue-wide polarity. Thus, symmetry breaking results from PAR-3-dependent intracellular polarization coupled to HMR-1-based tissue-level communication that occurs through a non-adhesive signaling role for HMR-1. Intestinal cells gain initial asymmetry from differential contact duration as homotypic contacts last longer than heterotypic contacts, thus providing stable platforms for LPC assembly and offering a logical and likely conserved framework for how internal epithelia with no obvious pre-existing asymmetries can polarize.

The protein deacetylase SIRT6 maintains cellular homeostasis through multiple pathways that include the deacetylation of histone H3 and repression of transcription. Prior work suggests that SIRT6 is associated with chromatin and can substantially reduce global levels of H3 acetylation, but how SIRT6 is able to accomplish this feat is unknown. Here, we describe an exquisitely tight interaction between SIRT6 and nucleosome core particles, in which a 2:1 enzyme:nucleosome complex assembles via asymmetric binding with distinct affinities. While both SIRT6 molecules associate with the acidic patch on the nucleosome, we find that the intrinsically disordered SIRT6 C-terminus promotes binding at the higher affinity site through recognition of nucleosomal DNA. Together, multivalent interactions couple productive binding to efficient deacetylation of histones on endogenous chromatin. Unique among histone deacetylases, SIRT6 possesses the intrinsic capacity to tightly interact with nucleosomes for efficient activity., SIRT6 plays essential roles in metabolism, tumor suppression, and DNA repair through the deacetylation of histone substrates. Here the authors use biophysical methods to investigate the molecular basis for SIRT6 interaction with the nucleosome core particle.

Summary Enzyme-catalyzed proximity labeling (PL) has emerged as a critical approach for identifying protein-protein proximity interactions in cells; however, PL techniques were not historically practical in living multicellular organisms due to technical limitations. Here, we present a protocol for applying PL to living C. elegans using the biotin ligase mutant enzyme TurboID. We demonstrated PL in a tissue-specific and region-specific manner by focusing on non-centrosomal MTOCs (ncMTOCs) of intestinal cells. This protocol is useful for targeted in vivo protein network profiling. For complete details on the use and execution of this protocol, please refer to Sanchez et al. (2021)., Graphical abstract, Highlights • TurboID enabled biotin-based proximity labeling protocol for C. elegans • Experimental design guidelines for proximity labeling in C. elegans • A step-by-step TurboID protocol from transgene design to protein identification, Enzyme-catalyzed proximity labeling (PL) has emerged as a critical approach for identifying protein-protein proximity interactions in cells; however, PL techniques were not historically practical in living multicellular organisms due to technical limitations. Here, we present a protocol for applying PL to living C. elegans using the biotin ligase mutant enzyme TurboID. We demonstrated PL in a tissue-specific and region-specific manner by focusing on non-centrosomal MTOCs (ncMTOCs) of intestinal cells. This protocol is useful for targeted in vivo protein network profiling.

SUMMARY 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., Graphical Abstract, In Brief Members of the Sirt6 family of histone deacetylases are known to prevent genomic instability and to regulate transcription. Feldman and Peterson find that yeast Sirt6 homologs repress transcription, preventing formation of excess RNA-DNA hybrids. Increased R loops are associated with new DNA double-strand breaks, linking transcriptional regulation to genomic stability.

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.

All living things are made up of cells that form the different tissues, organs and structures of an organism. The human body, for example, is thought to consist of some 37 trillion cells and harbor over 200 cell types. To maintain a working organism, cells divide to create new cells and replace the ones that have died. Cell division is a tightly controlled process consisting of several steps, and cells continuously face a Shakespearean dilemma of deciding whether to continue dividing (also known as cell proliferation) or to halt the process (known as quiescence). This difficult balancing act is critical during all stages of life, from embryonic development to tissue growth in an adult. Problems in the underlying pathways can result in diseases such as cancer. Cell division is driven by proteins called CDKs, which help cells to complete their cell cycle in the correct sequence. To gain more insight into this complex process, scientists have developed tools for monitoring CDKs. One such tool is a fluorescent biosensor, a molecule that can be inserted into cells that glows and moves in response to CDK activity. The biosensor can be studied and measured in each cell using a microscope. Adikes, Kohrman, Martinez et al. adapted and optimized an existing CDK biosensor to help study cell division and the switch between proliferation and quiescence in two common research organisms, the nematode Caenorhabditis elegans and the zebrafish. Analysis of this biosensor showed that CDK activity at the end of cell division is higher if the cells will divide again but is low if the cells are going to become quiescent. This could suggest that the decision of a cell between proliferation and quiescence may happen earlier than expected. The optimized biosensor is sensitive enough to detect these differences and can even measure variations that influence proliferation in a region on C. elegans that was once thought to be unchanging. The development of this biosensor provides a useful research tool that could be used in other living organisms. Many research questions relate to cell division and so the applications of this tool are wide ranging.

Summary Neurons are highly polarized cells with morphologically and functionally distinct dendritic and axonal processes. The molecular mechanisms that establish axon-dendrite polarity in vivo are poorly understood. Here, we describe the initial polarization of posterior deirid (PDE), a ciliated mechanosensory neuron, during development in vivo through 4D live imaging with endogenously tagged proteins. PDE inherits and maintains apicobasal polarity from its epithelial precursor. Its apical domain is directly transformed into the ciliated dendritic tip through apical constriction, which is followed by axonal outgrowth from the opposite basal side of the cell. The apical Par complex and junctional proteins persistently localize at the developing dendritic domain throughout this transition. Consistent with their instructive role in axon-dendrite polarization, conditional depletion of the Par complex and junctional proteins results in robust defects in dendrite and axon formation. During apical constriction, a microtubule-organizing center (MTOC) containing the microtubule nucleator γ-tubulin ring complex (γ-TuRC) forms along the apical junction between PDE and its sister cell in a manner dependent on the Par complex and junctional proteins. This junctional MTOC patterns neuronal microtubule polarity and facilitate the dynein-dependent recruitment of the basal body for ciliogenesis. When non-ciliated neurons are genetically manipulated to obtain ciliated neuronal fate, inherited apicobasal polarity is required for generating ciliated dendritic tips. We propose that inherited apicobasal polarity, together with apical cell-cell interactions drive the morphological and cytoskeletal polarity in early neuronal differentiation.

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 morphogenetic assaults. Using the developingC. elegansintestine as anin vivomodel, we investigated how epithelial cells maintain 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.

SummaryReorganization of microtubules from the centrosome to non-centrosomal subcellular sites is central to cell differentiation. To identify components of non-centrosomal microtubule organizing centers in differentiated cells of a living organism, we developed the biotin ligase-based proximity labeling approach TurboID for use inC. elegans. We identified proteins proximal to the non-centrosomal microtubule minus end protein PTRN-1/Patronin at the apical membrane of epithelial cells, focusing on two conserved proteins: spectraplakin protein VAB-10B and WDR-62, a protein we identify as homologous to vertebrate primary microcephaly disease gene WDR62. We found that WDR-62 and VAB-10B independently regulate the growth and localization of non-centrosomal microtubules and the apical targeting of microtubule minus end proteins. This regulation occurs downstream of cell polarity and in conjunction with actin. Our data suggest a division of labor where microtubule growth and anchoring are regulated by distinct complexes and uncover novel functions of spectraplakins and WDR62 family proteins.

Heterochromatin is a repressive, specialized chromatin structure that is central to eukaryotic transcriptional regulation and genome stability. In the budding yeast,Saccharomyces cerevisiae, heterochromatin formation requires Sir2p, Sir3p, and Sir4p, and these Sir proteins create specialized chromatin structures at telomeres and silent mating type loci. Previously, we reported that the SWI/SNF chromatin remodeling enzyme can evict Sir3 from chromatin fibersin vitro, though whether this activity contributes to the role of SWI/SNF as a transcriptional activator at euchromatic loci is unknown. Here, we characterize genetic interactions between theSIRgenes (SIR2,SIR3, andSIR4) and genes encoding subunits of the chromatin remodelers SWI/SNF and INO80C, as well genes encoding the histone deacetylases Hst3 and Hst4. We find that loss ofSIRgenes partially rescues the growth defects ofswi2,ino80, andhst3/hst4mutants during replication stress conditions. Interestingly, partial suppression ofswi2,ino80, andhst3 hst4mutant phenotypes is due to the pseudo-diploid state ofsirmutants, but a significant portion is due to more direct functional interactions. Consistent with this view, transcriptional profiling of strains lacking Swi2 or Sir3 identifies a set of genes whose expression in the M/G1 phase of the cell cycle requires SWI/SNF to antagonize the repressive impact of Sir3.

This scoping paper considers how digital tools, such as ICTs and AI, have failed to contribute to the “common good” in any sustained or scalable way. This is attributed to a problem that is at once political-economic and technical. Many digital tools’ business models are predicated on advertising: framing the user as an individual consumer-to-be-targeted, not as an organization, movement, or any sort of commons. At the level of infrastructure and hardware, the increased privatization and centralization of transmission and production leads to a dangerous bottlenecking of communication power, and to labor and production practices that are undemocratic and damaging to common resources. These practices escalate collective action problems, pose a threat to democratic decision making, aggravate issues of economic and labor inequality, and harm the environment and health. At the same time, the growth of both AI and online community formation raise questions around the very definition of human subjectivity and modes of relationality. Based on an operational definition of the common good grounded in ethics of care, sustainability, and redistributive justice, suggestions are made for solutions and further research in the areas of participatory design, digital democracy, digital labor, and environmental sustainability.

Microtubules are polarized intracellular polymers that play key roles in the cell including in transport, polarity, and cell division. Across eukaryotic cell types, microtubules adopt diverse intracellular organization to accommodate these distinct functions coordinated by specific cellular sites called microtubule organizing centers (MTOCs). Over 50 years of research on MTOC biology has focused mainly on the centrosome, however most differentiated cells employ non-centrosomal MTOCs (ncMTOCs) to organize their microtubules into diverse arrays which are critical to cell function. To identify essential ncMTOC components, we developed the biotin ligase-based proximity labeling approach TurboID for use in C. elegans. We identified proteins proximal to the microtubule minus end protein PTRN-1/Patronin at the apical ncMTOC of intestinal epithelial cells, focusing on two conserved proteins: spectraplakin protein VAB-10B and WDR-62, a protein we identify as homologous to vertebrate primary microcephaly disease gene WDR62. We found that WDR-62 and VAB-10B independently regulate the growth and localization of non-centrosomal microtubules and the apical targeting of microtubule minus end proteins. Depletion of VAB-10B resulted in disorganized microtubules and delayed localization of a microtubule nucleation complex γ-TuRC while loss of WDR-62 decreased the number of dynamic microtubules and abolished γ-TuRC localization. This regulation occurs downstream of cell polarity and in conjunction with actin. As this is the first report for non-centrosomal roles of WDR62 family proteins, our study expands the basic cell biological roles of this important disease protein. Together, our data suggest a division of labor where microtubule growth and anchoring are regulated by distinct complexes.

During mitosis in animal cells, the centrosome acts as a microtubule organizing center (MTOC) to assemble the mitotic spindle. MTOC function at the centrosome is driven by proteins within the pericentriolar material (PCM), however the molecular complexity of the PCM makes it difficult to differentiate the proteins required for MTOC activity from other centrosomal functions. We used the natural spatial separation of PCM proteins during mitotic exit to identify a minimal module of proteins required for centrosomal MTOC function in C. elegans. Using tissue specific degradation, we show that SPD-5, the functional homolog of CDK5RAP2, is essential for embryonic mitosis while SPD-2/CEP192 and PCMD-1, which are essential in the zygote, are dispensable. Surprisingly, although the centriole is known to be degraded in the ciliated sensory neurons in C. elegans , we find evidence for “centriole-less PCM” at the base of cilia and use this structure as a minimal testbed to dissect centrosomal MTOC function. Super-resolution imaging revealed that this PCM inserts inside the lumen of the ciliary axoneme and directly nucleates the assembly of dendritic microtubules towards the cell body. Tissue-specific degradation in ciliated sensory neurons revealed a role for SPD-5 and the conserved microtubule nucleator [[EQUATION]] -TuRC, but not SPD-2 or PCMD-1, in MTOC function at centriole-less PCM. This MTOC function was in the absence of regulation by mitotic kinases, highlighting the intrinsic ability of these proteins to drive microtubule growth and organization and further supporting a model that SPD-5 is the primary driver of MTOC function at the PCM.

Protein interaction networks and protein compartmentalization underlie all signaling and regulatory processes in cells. Enzyme-catalyzed proximity labeling (PL) has emerged as a new approach to study the spatial and interaction characteristics of proteins in living cells. However, current PL methods require over 18 hour labeling times or utilize chemicals with limited cell permeability or high toxicity. We used yeast display-based directed evolution to engineer two promiscuous mutants of biotin ligase, TurboID and miniTurbo, which catalyze PL with much greater efficiency than BioID or BioID2, and enable 10-minute PL in cells with non-toxic and easily deliverable biotin. Furthermore, TurboID extends biotin-based PL to flies and worms.

Summary Microtubules are polarized intracellular polymers that play key roles in the cell, including in transport, polarity, and cell division. Across eukaryotic cell types, microtubules adopt diverse intracellular organization to accommodate these distinct functions coordinated by specific cellular sites called microtubule-organizing centers (MTOCs). Over 50 years of research on MTOC biology has focused mainly on the centrosome; however, most differentiated cells employ non-centrosomal MTOCs (ncMTOCs) to organize their microtubules into diverse arrays, which are critical to cell function. To identify essential ncMTOC components, we developed the biotin ligase-based, proximity-labeling approach TurboID for use in C. elegans. We identified proteins proximal to the microtubule minus end protein PTRN-1/Patronin at the apical ncMTOC of intestinal epithelial cells, focusing on two conserved proteins: spectraplakin protein VAB-10B/MACF1 and WDR-62, a protein we identify as homologous to vertebrate primary microcephaly disease protein WDR62. VAB-10B and WDR-62 do not associate with the centrosome and instead specifically regulate non-centrosomal microtubules and the apical targeting of microtubule minus-end proteins. Depletion of VAB-10B resulted in microtubule mislocalization and delayed localization of a microtubule nucleation complex ɣ-tubulin ring complex (γ-TuRC), while loss of WDR-62 decreased the number of dynamic microtubules and abolished γ-TuRC localization. This regulation occurs downstream of cell polarity and in conjunction with actin. As this is the first report for non-centrosomal roles of WDR62 family proteins, we expand the basic cell biological roles of this important disease protein. Our studies identify essential ncMTOC components and suggest a division of labor where microtubule growth and localization are distinctly regulated.

SummaryCell proliferation and terminal differentiation are intimately coordinated during metazoan development. Here, we adapt a cyclin-dependent kinase (CDK) sensor to uncouple these cell cycle-associated events live inC. elegansand zebrafish. The CDK sensor consists of a fluorescently tagged CDK substrate that steadily translocates from the nucleus to the cytoplasm in response to increasing CDK activity and consequent sensor phosphorylation. We show that the CDK sensor can distinguish cycling cells in G1 from terminally differentiated cells in G0, revealing a commitment point and a cryptic stochasticity in an otherwise invariantC. eleganscell lineage. We also derive a predictive model of future proliferation behavior inC. elegansand zebrafish based on a snapshot of CDK activity in newly born cells. Thus, we introduce a live-cell imaging tool to facilitatein vivostudies of cell cycle control in a wide-range of developmental contexts.

A polarized arrangement of neuronal microtubule arrays is the foundation of membrane trafficking and subcellular compartmentalization. Conserved among both invertebrates and vertebrates, axons contain exclusively “plus-end-out” microtubules while dendrites contain a high percentage of “minus-end-out” microtubules, the origins of which have been a mystery. Here we show that the dendritic growth cone contains a non-centrosomal microtubule organizing center (ncMTOC), which generates minus-end-out microtubules along outgrowing dendrites and plus-end-out microtubules in the growth cone. RAB-11-positive recycling endosomes accumulate in this region and are responsible for localizing the microtubule nucleation complex γ-TuRC. The MTOC tracks the extending growth cone by kinesin-1/UNC-116-mediated endosome movements on distal plus-end-out microtubules and dynein-mediated endosome clustering near MTOC. Critically, perturbation of the function or localization of the MTOC causes reversed microtubule polarity in dendrites. These findings unveil the dendritic MTOC as a critical organelle for establishing axon-dendrite polarity.

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.

SummaryDuring mitosis in animal cells, the centrosome acts as a microtubule organizing center (MTOC) to assemble the mitotic spindle. MTOC function at the centrosome is driven by proteins within the pericentriolar material (PCM), however the molecular complexity of the PCM makes it difficult to differentiate the proteins required for MTOC activity from other centrosomal functions. We used the natural spatial separation of PCM proteins during mitotic exit to identify a minimal module of proteins required for centrosomal MTOC function in C. elegans. Using tissue specific degradation, we show that SPD-5, the functional homolog of CDK5RAP2, is essential for embryonic mitosis while SPD-2/CEP192 and PCMD-1, which are essential in the zygote, are dispensable. Surprisingly, although the centriole is known to be degraded in the ciliated sensory neurons in C. elegans [1-3], we find evidence for “centriole-less PCM” at the base of cilia and use this structure as a minimal testbed to dissect centrosomal MTOC function. Super-resolution imaging revealed that this PCM inserts inside the lumen of the ciliary axoneme and directly nucleates the assembly of dendritic microtubules towards the cell body. Tissue-specific degradation in ciliated sensory neurons revealed a role for SPD-5 and the conserved microtubule nucleator γ-TuRC, but not SPD-2 or PCMD-1, in MTOC function at centriole-less PCM. This MTOC function was in the absence of regulation by mitotic kinases, highlighting the intrinsic ability of these proteins to drive microtubule growth and organization and further supporting a model that SPD-5 is the primary driver of MTOC function at the PCM.

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., Author summary Eukaryotic cells require specific arrangements of microtubules to carry out diverse functions, including cell division and intracellular transport. In dividing animal cells, microtubules are arranged radially around two centrosomes, promoting the correct distribution of DNA into daughter cells. As cells differentiate, microtubules become organized at non-centrosomal sites, often yielding decentralized microtubule arrays. Although a large body of work has focused on understanding how microtubules are grown from and organized by the centrosome, very little is known about how these activities are performed at non-centrosomal sites. We optimized a technique to degrade proteins in C. elegans differentiated cells, specifically testing the role of essential centrosome proteins in building non-centrosomal microtubules, which emanate from the apical membrane in embryonic intestinal epithelial cells. Surprisingly, we found fundamental differences between the centrosome and membrane, both in the mechanisms that recruit microtubule regulators to each site, and in the proteins that are required to build and organize microtubules. In addition, we found that when fewer microtubules grew from membranes, the remaining microtubules grew faster, suggesting competition for limited materials. Together, our study highlights differences in how centrosomes and membranes grow and organize microtubules, and that multiple pathways contribute membrane-organized microtubules.

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 co-substrates 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.

Protein interaction networks and protein compartmentation underlie every signaling process and regulatory mechanism in cells. Recently, proximity labeling (PL) has emerged as a new approach to study the spatial and interaction characteristics of proteins in living cells. However, the two enzymes commonly used for PL come with tradeoffs – BioID is slow, requiring tagging times of 18-24 hours, while APEX peroxidase uses substrates that have limited cell permeability and high toxicity. To address these problems, we used yeast display-based directed evolution to engineer two mutants of biotin ligase, TurboID and miniTurbo, with much greater catalytic efficiency than BioID, and the ability to carry out PL in cells in much shorter time windows (as little as 10 minutes) with non-toxic and easily deliverable biotin. In addition to shortening PL time by 100-fold and increasing PL yield in cell culture, TurboID enabled biotin-based PL in new settings, including yeast, Drosophila, and C. elegans.

A mutation in a microtubule-severing enzyme, katanin, causes flagella to become short due to a reduced cytoplasmic precursor pool. These results suggest that competition between flagella and cytoplasmic microtubules for a limited tubulin pool is facilitated by katanin, which is confirmed by stochastic models., 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.

Genetic code expansion has provided the ability to site-specifically incorporate a multitude of noncanonical amino acids (ncAAs) into proteins for a wide variety of applications, but low ncAA incorporation efficiency can hamper the utility of this powerful technology. When investigating proteins containing the post-translational modification 3-nitro-tyrosine (nitroTyr), we developed second-generation amino-acyl tRNA synthetases (RS) that incorporate nitroTyr at efficiencies roughly an order of magnitude greater than those previously reported and that advanced our ability to elucidate the role of elevated cellular nitroTyr levels in human disease (e.g., Franco, M. et al. Proc. Natl. Acad. Sci. U.S.A2013, 110, E110223487751). Here, we explore the origins of the improvement achieved in these second-generation RSs. Crystal structures of the most efficient of these synthetases reveal the molecular basis for the enhanced efficiencies observed in the second-generation nitroTyr-RSs. Although Tyr is not detectably incorporated into proteins when expression media is supplemented with 1 mM nitroTyr, a major difference between the first- and second-generation RSs is that the second-generation RSs have an active site more compatible with Tyr binding. This feature of the second-generation nitroTyr-RSs appears to be the result of using less stringent criteria when selecting from a library of mutants. The observation that a different selection strategy performed on the same library of mutants produced nitroTyr-RSs with dramatically improved efficiencies suggests the optimization of established selection protocols could lead to notable improvements in ncAA-RS efficiencies and thus the overall utility of this technology.

Mammalian sirtuins (SIRT1 through SIRT7) are members of a highly conserved family of NAD+-dependent protein deacetylases that function in metabolism, genome maintenance, and stress responses. Emerging evidence suggests that some sirtuins display substrate specificity toward other acyl groups attached to the lysine ϵ-amine. SIRT6 was recently reported to preferentially hydrolyze long-chain fatty acyl groups over acetyl groups. Here we investigated the catalytic ability of all sirtuins to hydrolyze 13 different acyl groups from histone H3 peptides, ranging in carbon length, saturation, and chemical diversity. We find that long-chain deacylation is a general feature of mammalian sirtuins, that SIRT1 and SIRT2 act as efficient decrotonylases, and that SIRT1, SIRT2, SIRT3, and SIRT4 can remove lipoic acid. These results provide new insight into sirtuin function and a means for cellular removal of an expanding list of endogenous lysine modifications. Given that SIRT6 is a poor deacetylase in vitro, but binds and prefers to hydrolyze long-chain acylated peptides, we hypothesize that binding of certain free fatty acids (FFAs) could stimulate deacetylation activity. Indeed, we demonstrate that several biologically relevant FFAs (including myristic, oleic, and linoleic acids) at physiological concentrations induce up to a 35-fold increase in catalytic efficiency of SIRT6 but not SIRT1. The activation mechanism is consistent with fatty acid inducing a conformation that binds acetylated H3 with greater affinity. Binding of long-chain FFA and myristoylated H3 peptide is mutually exclusive. We discuss the implications of discovering endogenous, small-molecule activators of SIRT6. Background: Sirtuins regulate metabolism, genome maintenance, and stress responses. Results: Long-chain free fatty acids stimulate SIRT6 deacetylase, and sirtuins display distinct but overlapping specificity for diverse acylated peptides. Conclusion: SIRT6 is activated by biologically relevant fatty acids, and long-chain deacylation is a general feature of sirtuins. Significance: Discovery of endogenous, small-molecule activators of SIRT6 demonstrates the therapeutic potential of compounds that promote SIRT6 function.

Sirtuins are a family of NAD(+)-dependent protein deacetylases/deacylases that dynamically regulate transcription, metabolism, and cellular stress response. Their general positive link with improved health span in mammals, potential regulation of pathways mediated by caloric restriction, and growing links to human disease have spurred interest in therapeutics that target their functions. Here, we review the current understanding of the chemistry of catalysis, biological targets, and endogenous regulation of sirtuin activity. We discuss recent efforts to generate small-molecule regulators of sirtuin activity.

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.

Cilia are motile and sensory organelles with critical roles in physiology. Ciliary defects can cause numerous human disease symptoms including polycystic kidneys, hydrocephalus, and retinal degeneration. Despite the importance of these organelles, their assembly and function is not fully understood. The unicellular green alga Chlamydomonas reinhardtii has many advantages as a model system for studies of ciliary assembly and function. Here we describe our initial efforts to build a chemical-biology toolkit to augment the genetic tools available for studying cilia in this organism, with the goal of being able to reversibly perturb ciliary function on a rapid time-scale compared to that available with traditional genetic methods. We screened a set of 5520 compounds from which we identified four candidate compounds with reproducible effects on flagella at nontoxic doses. Three of these compounds resulted in flagellar paralysis and one induced flagellar shortening in a reversible and dose-dependent fashion, accompanied by a reduction in the speed of intraflagellar transport. This latter compound also reduced the length of cilia in mammalian cells, hence we named the compound “ciliabrevin” due to its ability to shorten cilia. This compound also robustly and reversibly inhibited microtubule movement and retrograde actin flow in Drosophila S2 cells. Ciliabrevin may prove especially useful for the study of retrograde actin flow at the leading edge of cells, as it slows the retrograde flow in a tunable dose-dependent fashion until flow completely stops at high concentrations, and these effects are quickly reversed upon washout of the drug. © 2011 Wiley-Liss, Inc.

An intriguing feature of centrioles is that these highly complicated microtubule-based structures duplicate once per cell cycle and the cell has precise control over their number. Each cell contains exactly two centrioles, linked together as a pair, one of which is a mother centriole formed in a previous cell cycle and the other a daughter centriole whose assembly is templated by the mother. Neither the molecular basis nor the functional role of mother-daughter centriole linkage is understood. We have identified a mutant, asq2, with defects in centriole linkage. asq2 mutant cells have variable numbers of centrioles and defects in centriole positioning. Here, we show that ASQ2 encodes the novel conserved protein, TBCCd1, a member of a protein family that includes a tubulin folding co-chaperone and the retinitis pigmentosa protein, RP2, involved in tubulin quality control during ciliogenesis. We characterize mitosis in asq2 cells. We show that the majority of cells establish a bipolar spindle, but that cells have defects in spindle orientation. A small subset of asq2 cells have centrioles at both poles, and these cells have properly positioned spindles, indicating that centrioles at the poles may be important for spindle orientation. The defects in centriole number control, centriole positioning, and spindle orientation appear to arise from a primary defect in centriole linkage mediated by TBCCd1/ASQ2.

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. Fil: Kugel, Sita . Harvard Medical School; Estados Unidos Fil: Feldman, Jessica L. . University Of Wisconsin; Estados Unidos Fil: Klein, Mark A.. University Of Wisconsin; Estados Unidos Fil: Silberman, Dafne Magali. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; Argentina. Universidad de Buenos Aires. Facultad de Medicina; Argentina Fil: Sebastián, Carlos. Harvard Medical School; Estados Unidos Fil: Mermel, Craig. Harvard Medical School; Estados Unidos Fil: Dobersch, Stephanie . Institute Max Planck for Heart and Lung Research; Alemania Fil: Clark, Abbe R.. Harvard Medical School; Estados Unidos Fil: Getz, Gad. Harvard Medical School; Estados Unidos Fil: Denu, John M.. University Of Wisconsin; Estados Unidos Fil: Mostoslavsky, Raul. Harvard Medical School; Estados Unidos

Precise temporal and spatial control of transcription is a fundamental component of embryonic development. Regulation of transcription elongation can act as a rate-limiting step during mRNA synthesis. The mechanisms of stimulation and repression of transcription elongation during development are not yet understood. We have identified a class of zebrafish mutations (pandora, sk8 and s30) that cause multiple developmental defects, including discrete problems with pigmentation, tail outgrowth, ear formation and cardiac differentiation. We demonstrate that the pandora gene encodes a protein similar to Spt6, a proposed transcription elongation factor. Additionally, the sk8 and s30 mutations are null alleles of the foggy/spt5 locus, which encodes another transcription elongation factor. Through real-time RT-PCR analysis, we demonstrate that Spt6 and Spt5 are both required for efficient kinetics of hsp70 transcription in vivo. Altogether, our results suggest that Spt6 and Spt5 play essential roles of comparable importance for promoting transcription during embryogenesis. This study provides the first genetic evidence for parallel functions of Spt6 and Spt5 in metazoans and establishes a system for the future analysis of transcription elongation during development.Supplemental figure available on-line