Faculty Bibliography

Background/Purpose: We aimed to understand the mechanism by which membrane-type 1 matrix metalloproteinase (MT1-MMP, MMP-14) controls bone and cartilage homeostasis. MT1-MMP, a cell-membrane-bound proteinase with an extracellular catalytic site and a 20-amino acid cytoplasmic tail, plays a key role in postnatal bone formation. The genetic deficiency of MT1-MMP in the mouse causes dwarfism, osteopenia and severe arthritis. Deletion of MT1-MMP in bone marrow-derived mesenchymal progenitor cells (BM-MSC) recapitulates this phenotype, showing that MT1-MMP controls osteogenic differentiation in MSC. The phenotype of MT1-MMP-/- mice has been proposed to result from lack of MT1-MMP proteolytic activity. However, mounting evidence shows a variety of proteolysis-independent signaling functions of MT1-MMP. The unique tyrosine (Y573) in the MT1-MMP cytoplasmic tail is fundamental for the control of intracellular signaling. Methods: We generated a mouse with the Y573D mutation in MT1-MMP (MT1-MMP Y573D) and characterized its skeletal phenotype by histological and microCT analyses. Isolated BM-MSC were induced to differentiate into osteoblasts, chondrocytes and adipocytes, using qRT-PCR to analyze gene expression. Mouse C3H10T1/2 MSC were transfected with MT1-MMP cDNA and analyzed for Wnt signaling by luciferase reporter assays. Results: MT1-MMP Y573D mice had increased trabecular bone relative to wt littermates, marked thinning of articular cartilage with disorganized tissue architecture, clustering and cloning of chondrocytes, and pronounced decrease in bone marrow-associated and total body fat. We induced BM-MSC from wt and MT1-MMP Y573D littermates to differentiate into osteoblast and chondrocytes, and myeloid precursors into osteoclasts. The Y573D mutation dramatically increased MSC expression of osteoblast markers and strongly downregulated chondrocyte and osteoclast markers. These findings indicated that Wnt signaling is upregulated in MT1-MMP Y573D-expressing MSC. Therefore, we analyzed Wnt signaling. We transiently transfected C3H10T1/2 MSC cells in osteoblast medium with the cDNAs for wt MT1-MMP and MT1-MMP Y573D. As controls the cells were transfected with the empty vector (pcDNA) or with MT1-MMP E240A, a mutant devoid of proteolytic activity. MT1-MMP Y573D dramatically upregulated Wnt signaling relative to wt MT1-MMP and MT1-MMP E240A. Conclusion: MT1-MMP controls Wnt signaling by a mechanism independent of extracellular proteolysis and mediated by its cytoplasmic tail. MT1-MMP is a bifunctional protein, with an extracellular proteolytic activity that promotes bone formation through ECM remodeling and a cytoplasmic tail that controls osteogenesis by interacting with a key pro-osteogenic signaling pathway

Recent developments in tissue clearing methods have provided investigators with an invaluable tool for visualizing and mapping three dimensional macromolecular structures and processes. By implementing a routine protocol, based on the passive clarity technique (PACT) method, for tissue clearing, the Research Histopathology Core at NYU Langone Medical Center seeks to provide investigators with a reliable, customizable service in conjunction with the immunohistochemistry (IHC) and Microscopy Cores in order to produce results in the most efficient way possible for both the investigators and the cores. The PACT method of clearing allows visualization of endogenous fluorescence and immunofluorescence labeling performed by the Core, or both. Stabilization through transparent hydrogel cross-linking, followed by delipidation in an sodium dodecyl sulfate buffer, results in a clear tissue sample that remains structurally sound with proteins, nucleic acids, and any associated labels in place. The clearing buffer can also be modified to allow simultaneous decalcification of bone specimens. Final clearing is achieved in a refractive index matching solution (RIMS buffer) which also serves as the microscopy medium. Cleared and labeled tissue can then be imaged on the Microscopy Core's Zeiss lightsheet microscope, allowing multichannel fluorescence from a range of angles and Z-stacking. The lightsheet microscope excites and detects only one thin optical section of the specimen at a time, making three dimensional imaging exceptionally light efficient. By honing proficiency in tissue clearing via the PACT method and working in close collaboration with neighboring core labs, the Histopathology Core can increase its breadth of expertise while relieving investigators of the time and cost intensive burden of protocol development and training.

Reticulon-4 (RTN4) (also called Nogo-A/B) and the atlastin (ATL) GTPases have critical roles in the formation of tubules and sheets of the standard peripheral endoplasmic reticulum (ER) in mammalian cells. The interferon-inducible protein MxA (myxovirus resistance protein A) is a dynamin-family, atlastin-like GTPase with membrane-binding and tubulation activity. While MxA has been investigated extensively for its antiviral effects, less is known about its role in cellular physiology in the uninfected cell. Over the last 15 years MxA has been represented to localize to "subcompartments of the smooth ER." However these prior studies did not include ER structural proteins as markers. In contrast, using RTN4 as an ER marker, we discovered that MxA expressed in human cell lines associated with large tubuloreticular structures that were distinct from the standard RTN4/ATL3-based ER. Our methods included immunofluorescence studies, thin-section EM, and single and double-label immunoEM. MxA generated large variably sized tubuloreticular structures that were RTN-4 negative. In contrast to HAtagged ATL3 which colocalized with RTN4 and increased ER sheets at three-way junctions, HA-tagged MxA tubules were independent of RTN4 and MxA did not affect the standard RTN4-based ER. Thus, remarkably, one and the same cell contained two distinct tubuloreticular systems - one based on RTN4/ATL3 and the other on MxA. The MxA-reticulum is a novel organelle which is positive for EEA1, clathrin light chain, Rab5, Rab11 and GRP78/BiP, but not for LAMP2, Rab7 and syntaxin 17. The present studies advance the novel paradigm that different atlastin-like GTPases can generate distinct ER-like tubuloreticular systems within the same cell. The new data require reinterpretion of prior studies in the MxA field over the last decade

The process of embryonic morphogenesis involves cell shape changes, which are accompanied by constant remodeling of cell junctions. Rho GTPases are signaling proteins that regulate adherens junctions (AJ) by mediating the recycling of junction components, actin polymerization, and myosin activity. We investigated the role of the CDC-42 GTPase during elongation of C. elegans embryo, during which epidermal cells extend along their anterior-posterior axis and shrink along their dorsal-ventral axis, lengthening the embryo four-fold. Depletion of CDC-42 caused embryonic lethality at different stages of elongation. Overactivation of CDC-42 in sensitized hmp-1/alpha-catenin hypomorphic mutants, which have partially compromised junctions, also led to embryonic arrest, suggesting that CDC-42 activity might be regulated at junctions during elongation. Rho GTPases are activated by GEFs and inhibited by RhoGAPs. Looking for a possible regulator of CDC-42 at the junctions, we found that the conserved C. elegans RhoGAP PAC-1/ARHGAP21 co-localizes with CDC-42 at adherens junctions in embryonic epithelial tissues. Genetic interactions tests showed that PICC-1/CCDC85A-C, which encodes a coiled-coil protein that interacts with PAC-1 and with the junction component JAC-1/p120, likely functions with PAC-1 in the same pathway during elongation. pac-1 mutant embryos develop normally, but pac-1 mutations enhance the lethality of hypomorphic hmp-1 mutants, as we observed in the case of CDC-42 overactivation. The RhoGAP activity of PAC-1 is required for its function, but not for its localization, suggesting that PAC-1 regulates the strength of AJs by locally inhibiting CDC-42 GTPase activity. In support of this hypothesis, decreasing CDC-42 levels partially rescues the lethality of hmp-1 pac-1 double mutants, and overexpressing PAC-1 lowers levels of an active CDC-42 biosensor at AJs. In pac-1 mutants and embryos expressing constitutively active CDC-42, levels of AJ proteins were increased at junctions. Consistent with this increase being the cause of elongation defects, overexpression of HMR-1/ E-cadherin enhances the lethality of hmp-1 mutants. Based on these results, we propose that CDC-42 activity must be limited at junctions to ensure that appropriate levels of junction components are present as these structures remodel during morphogenetic events

The genomic evolution inherent to cancer relates directly to a renewed focus on the voluminous next-generation sequencing data and machine learning for the inference of explanatory models of how the (epi)genomic events are choreographed in cancer initiation and development. However, despite the increasing availability of multiple additional -omics data, this quest has been frustrated by various theoretical and technical hurdles, mostly stemming from the dramatic heterogeneity of the disease. In this paper, we build on our recent work on the "selective advantage" relation among driver mutations in cancer progression and investigate its applicability to the modeling problem at the population level. Here, we introduce PiCnIc (Pipeline for Cancer Inference), a versatile, modular, and customizable pipeline to extract ensemble-level progression models from cross-sectional sequenced cancer genomes. The pipeline has many translational implications because it combines state-of-the-art techniques for sample stratification, driver selection, identification of fitness-equivalent exclusive alterations, and progression model inference. We demonstrate PiCnIc's ability to reproduce much of the current knowledge on colorectal cancer progression as well as to suggest novel experimentally verifiable hypotheses.

BACKGROUND: Colorectal cancer is a heterogeneous disease arising from at least two precursors-the conventional adenoma (CA) and the serrated polyp. We and others have previously shown a relationship between the human gut microbiota and colorectal cancer; however, its relationship to the different early precursors of colorectal cancer is understudied. We tested, for the first time, the relationship of the gut microbiota to specific colorectal polyp types. RESULTS: Gut microbiota were assessed in 540 colonoscopy-screened adults by 16S rRNA gene sequencing of stool samples. Participants were categorized as CA cases (n = 144), serrated polyp cases (n = 73), or polyp-free controls (n = 323). CA cases were further classified as proximal (n = 87) or distal (n = 55) and as non-advanced (n = 121) or advanced (n = 22). Serrated polyp cases were further classified as hyperplastic polyp (HP; n = 40) or sessile serrated adenoma (SSA; n = 33). We compared gut microbiota diversity, overall composition, and normalized taxon abundance among these groups. CA cases had lower species richness in stool than controls (p = 0.03); in particular, this association was strongest for advanced CA cases (p = 0.004). In relation to overall microbiota composition, only distal or advanced CA cases differed significantly from controls (p = 0.02 and p = 0.002). In taxon-based analysis, stool of CA cases was depleted in a network of Clostridia operational taxonomic units from families Ruminococcaceae, Clostridiaceae, and Lachnospiraceae, and enriched in the classes Bacilli and Gammaproteobacteria, order Enterobacteriales, and genera Actinomyces and Streptococcus (all q < 0.10). SSA and HP cases did not differ in diversity or composition from controls, though sample size for these groups was small. Few taxa were differentially abundant between HP cases or SSA cases and controls; among them, class Erysipelotrichi was depleted in SSA cases. CONCLUSIONS: Our results indicate that gut microbes may play a role in the early stages of colorectal carcinogenesis through the development of CAs. Findings may have implications for developing colorectal cancer prevention therapies targeting early microbial drivers of colorectal carcinogenesis.

Although messenger RNA (mRNA) translation is a fundamental biological process, it has never been imaged in real time in vivo with single-molecule precision. To achieve this, we developed nascent chain tracking (NCT), a technique that uses multi-epitope tags and antibody-based fluorescent probes to quantify protein synthesis dynamics at the single-mRNA level. NCT reveals an elongation rate of ~10 amino acids per second, with initiation occurring stochastically every ~30 seconds. Polysomes contain ~1 ribosome every 200 to 900 nucleotides and are globular rather than elongated in shape. By developing multicolor probes, we showed that most polysomes act independently; however, a small fraction (~5%) form complexes in which two distinct mRNAs can be translated simultaneously. The sensitivity and versatility of NCT make it a powerful new tool for quantifying mRNA translation kinetics.

Protein clustering is a hallmark of genome regulation in mammalian cells. However, the dynamic molecular processes involved make it difficult to correlate clustering with functional consequences in vivo. We developed a live-cell super-resolution approach to uncover the correlation between mRNA synthesis and the dynamics of RNA Polymerase II (Pol II) clusters at a gene locus. For endogenous beta-actin genes in mouse embryonic fibroblasts, we observe that short-lived (~8 s) Pol II clusters correlate with basal mRNA output. During serum stimulation, a stereotyped increase in Pol II cluster lifetime correlates with a proportionate increase in the number of mRNAs synthesized. Our findings suggest that transient clustering of Pol II may constitute a pre-transcriptional regulatory event that predictably modulates nascent mRNA output.

Multifocus microscopy (MFM) allows high-resolution instantaneous three-dimensional (3D) imaging and has been applied to study biological specimens ranging from single molecules inside cells nuclei to entire embryos. We here describe pattern designs and nanofabrication methods for diffractive optics that optimize the light-efficiency of the central optical component of MFM: the diffractive multifocus grating (MFG). We also implement a "precise color" MFM layout with MFGs tailored to individual fluorophores in separate optical arms. The reported advancements enable faster and brighter volumetric time-lapse imaging of biological samples. In live microscopy applications, photon budget is a critical parameter and light-efficiency must be optimized to obtain the fastest possible frame rate while minimizing photodamage. We provide comprehensive descriptions and code for designing diffractive optical devices, and a detailed methods description for nanofabrication of devices. Theoretical efficiencies of reported designs is approximately 90% and we have obtained efficiencies of > 80% in MFGs of our own manufacture. We demonstrate the performance of a multi-phase MFG in 3D functional neuronal imaging in living C. elegans.

Small-molecule fluorophores are important tools for advanced imaging experiments. We previously reported a general method to improve small, cell-permeable fluorophores which resulted in the azetidine-containing 'Janelia Fluor' (JF) dyes. Here, we refine and extend the utility of these dyes by synthesizing photoactivatable derivatives that are compatible with live-cell labeling strategies. Once activated, these derived compounds retain the superior brightness and photostability of the JF dyes, enabling improved single-particle tracking and facile localization microscopy experiments.