The rockefeller university

We have developed a 3D super-resolution microscopy method that enables deep imaging in cells. This technique relies on the effective combination of multifocus microscopy and astigmatic 3D single-molecule localization microscopy. We describe the optical system and the fabrication process of its key element, the multifocus grating. Then, two strategies for localizing emitters with our imaging method are presented and compared with a previously described deep 3D localization algorithm. Finally, we demonstrate the performance of the method by imaging the nuclear envelope of eukaryotic cells reaching a depth of field of similar to 4 mu m. (C) 2016 Optical Society of America

Developmental specification of germ cells lies at the heart of inheritance, as germ cells contain all of the genetic and epigenetic information transmitted between generations. The critical developmental event distinguishing germline from somatic lineages is the differentiation of primordial germ cells (PGCs)(1,2), precursors of sex-specific gametes that produce an entire organism upon fertilization. Germ cells toggle between uni- and pluripotent states as they exhibit their own 'latent' form of pluripotency. For example, PGCs express a number of transcription factors in common with embryonic stem (ES) cells, including OCT4 (encoded by Pou5f1), SOX2, NANOG and PRDM14 (refs 2-4). A biochemical mechanism by which these transcription factors converge on chromatin to produce the dramatic rearrangements underlying ES-cell-and PGC-specific transcriptional programs remains poorly understood. Here we identify a novel co-repressor protein, CBFA2T2, that regulates pluripotency and germline specification in mice. Cbfa2t2(-/-) mice display severe defects in PGC maturation and epigenetic reprogramming. CBFA2T2 forms a biochemical complex with PRDM14, a germline-specific transcription factor. Mechanistically, CBFA2T2 oligomerizes to form a scaffold upon which PRDM14 and OCT4 are stabilized on chromatin. Thus, in contrast to the traditional 'passenger' role of a co-repressor, CBFA2T2 functions synergistically with transcription factors at the crossroads of the fundamental developmental plasticity between uni- and pluripotency.

While engagement of the inhibitory Fc gamma-receptor (Fc gamma R) IIB is an absolute requirement for in vivo antitumor activity of agonistic mouse anti-CD40 monoclonal antibodies (mAbs), a similar requirement for human mAbs has been disputed. By using a mouse model humanized for its Fc gamma Rs and CD40, we revealed that Fc gamma RIIB engagement is essential for the activity of human CD40 mAbs, while engagement of the activating Fc gamma RIIA inhibits this activity. By engineering Fc variants with selective enhanced binding to Fc gamma RIIB, but not to Fc gamma RIIA, significantly improved antitumor immunity was observed. These findings highlight the necessity of optimizing the Fc domain for this class of therapeutic antibodies by using appropriate preclinical models that accurately reflect the unique affinities and cellular expression of human Fc gamma R.

During the 1980s, the North Sea plankton community underwent a well-documented ecosystem regime shift, including both spatial changes (northward species range shifts) and temporal changes (increases in the total abundances of warmer water species). This regime shift has been attributed to climate change. Plankton provide a link between climate and higher trophic-level organisms, which can forage on large spatial and temporal scales. It is therefore important to understand not only whether climate change affects purely spatial or temporal aspects of plankton dynamics, but also whether it affects spatiotemporal aspects such as metapopulation synchrony. If plankton synchrony is altered, higher trophic-level feeding patterns may be modified. A second motivation for investigating changes in synchrony is that the possibility of such alterations has been examined for few organisms, in spite of the fact that synchrony is ubiquitous and of major importance in ecology. This study uses correlation coefficients and spectral analysis to investigate whether synchrony changed between the periods 1959-1980 and 1989-2010. Twenty-three plankton taxa, sea surface temperature (SST), and wind speed were examined. Results revealed that synchrony in SST and plankton was altered. Changes were idiosyncratic, and were not explained by changes in abundance. Changes in the synchrony of Calanus helgolandicus and Para-pseudocalanus spp appeared to be driven by changes in SST synchrony. This study is one of few to document alterations of synchrony and climate-change impacts on synchrony. We discuss why climate-change impacts on synchrony may well be more common and consequential than previously recognized.

Cholesterol homeostasis is mediated by Scap, a polytopic endoplasmic reticulum (ER) protein that transports sterol regulatory element-binding proteins from the ER to Golgi, where they are processed to forms that activate cholesterol synthesis. Scap has eight transmembrane helices and two large luminal loops, designated Loop1 and Loop7. We earlier provided indirect evidence that Loop1 binds to Loop7, allowing Scap to bind COPII proteins for transport in coated vesicles. When ER cholesterol rises, it binds to Loop1. We hypothesized that this causes dissociation from Loop7, abrogating COPII binding. Here we demonstrate direct binding of the two loops when expressed as isolated fragments or as a fusion protein. Expressed alone, Loop1 remained intracellular and membrane-bound. When Loop7 was co-expressed, it bound to Loop1, and the soluble complex was secreted. A Loop1-Loop7 fusion protein was also secreted, and the two loops remained bound when the linker between them was cleaved by a protease. Point mutations that disrupt the Loop1-Loop7 interaction prevented secretion of the Loop1-Loop7 fusion protein. These data provide direct documentation of intramolecular Loop1-Loop7 binding, a central event in cholesterol homeostasis.

The importance of TANK binding kinase-1 (TBK1), a multimeric kinase that modulates inflammation and autophagy, in human health has been highlighted for the first time by the recent discoveries of mutations in TBK1 that underlie amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), normal tension glaucoma (NTG) or childhood herpes simplex encephalitis (HSE). Gain-of-function of TBK1 are associated with NTG, whereas loss-of-function mutations result in ALS/FTD or in HSE. In light of these new findings, we review the role of TBK1 in these seemingly unrelated, yet allelic diseases, and discuss the role of TBK1 in neuroinflammatory diseases. This discovery has the potential to significantly increase our understanding of the molecular basis of these poorly understood diseases.

Despite abundant data supporting c-Src as a metastasis-promoting oncogene, activating mutations of c-Src are rare. This suggests that trans-interacting proteins may have a critical role in regulating c-Src activation. Here, we first report the discovery of Src homology 3 (SH3) domain-binding glutamic acid-rich-like protein (SH3BGRL), a novel c-Src activator in mice. Ectopic expression of murine SH3BGRL (mSH3BGRL) strongly promoted both tumor cell invasion and lung metastasis. Molecularly, mSH3BGRL specifically bound the inactive form of c-Src phosphorylated at Tyr527, promoting Tyr416 phosphorylation of c-Src and subsequent FAK-mediated activation of ERK and AKT signaling pathways. Targeting endogenous c-Src alone was sufficient to abolish mSH3BGRL-induced cancer metastasis in vivo. Unexpectedly, human SH3BGRL (hSH3BGRL) in turn suppressed tumorigenesis and metastasis in nature. We attempted site-specific reversion of hSH3BGRL amino-acid sequence to mSH3BGRL and found V108A substitution sufficient to restore SH3BGRL function as a c-Src activator and metastasis promoter. Notably, the somatic mutation R76C of hSH3BGRL can similarly act as hSH3BGRL-V108A and mSH3BGRL in tumorigenesis and metastasis. Our results uncover an evolutionarily controversial role of SH3BGRL in driving tumor metastasis through c-Src activation, and suggests that hSH3BGRL mutation status could be relevant to cancer diagnosis and therapy.

Transfer RNAs (tRNAs) are primarily viewed as static contributors to gene expression. By developing a high-throughput tRNA profiling method, we find that specific tRNAs are upregulated in human breast cancer cells as they gain metastatic activity. Through loss-of-function, gain-of-function, and clinical-association studies, we implicate tRNAGluUUC and tRNAArgCCG as promoters of breast cancer metastasis. Upregulation of these tRNAs enhances stability and ribosome occupancy of transcripts enriched for their cognate codons. Specifically, tRNAGluUUC promotes metastatic progression by directly enhancing EXOSC2 expression and enhancing GRIPAP1-constituting an "inducible" pathway driven by a tRNA. The cellular proteomic shift toward a prometastatic state mirrors global tRNA shifts, allowing for cell-state and cell-type transgene expression optimization through codon content quantification. TRNA modulation represents a mechanism by which cells achieve altered expression of specific transcripts and proteins. TRNAs are thus dynamic regulators of gene expression and the tRNA codon landscape can causally and specifically impact disease progression.

Information encoded in DNA is interpreted, modified, and propagated as chromatin. The diversity of inputs encountered by eukaryotic genomes demands a matching capacity for transcriptional outcomes provided by the combinatorial and dynamic nature of epigenetic processes. Advances in genome editing, visualization technology, and genome-wide analyses have revealed unprecedented complexity of chromatin pathways, offering explanations to long-standing questions and presenting new challenges. Here, we review recent findings, exemplified by the emerging understanding of crossregulatory interactions within chromatin, and emphasize the pathologic outcomes of epigenetic misregulation in cancer.