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Enabling Nanoscale Dynamic Imaging of Vesicles and Organelles

项目来源

美国卫生和人类服务部基金(HHS)

项目主持人

SAMMAK, PAUL J

项目受资助机构

YALE UNIVERSITY

项目编号

5R01GM118486-04

立项年度

2019

立项时间

未公开

研究期限

未知 / 未知

项目级别

国家级

受资助金额

837500.00美元

学科

Bioengineering; Biotechnology; Nanotechnology;

学科代码

未公开

基金类别

Non-SBIR/STTR RPGs

关键词

未公开

参与者

BADDELEY, DAVID ; BEWERSDORF, JOERG ; TOOMRE, DEREK K.

参与机构

NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

项目标书摘要：? DESCRIPTION (provided by applicant): The long-term aim of this project is to enable the imaging of vesicle and organelle dynamics inside living cells with unprecedented spatial and temporal resolution. The most compelling advantage of new super-resolution techniques such as single molecule switching (SMS) nanoscopy is the potential to image dynamic processes in living cells with 10-20 nm resolution and hence solve the many open questions in cell biology which need both high structural and temporal resolution. One such problem is how the Golgi is dynamically organized, a major and highly debated enigma. Although SMS imaging in fixed cells is already yielding impressive new biological discoveries, the potential to resolve dynamics is far from fully realized. Key limiting factors include: (i) lack of instrumentation capable of both attaining the highest resolutions and doing so in an environment and at a speed which are compatible with extended imaging in living cells, (ii) lack of good probes which can non-toxically label and switch inside a live cell with high specificity, density and brightness, and (iii) uncertainty about how to deal with the potentially incomplete data that high-speed super-resolution microscopy delivers. Motivated by a long-standing biological problem, namely the mechanism by which proteins are trafficked through the Golgi complex, we propose to address these major current limitations and develop the microscope hardware, probes and algorithms to make dynamic nanoscopy a reality. Our specific aims are: 1) To implement a 4Pi-SMS instrument which will deliver the best possible 3D resolution in living cells with minimal photodamage, 2) To develop a new genre of blinkable high-density live-cell SMS probes to image the Golgi, and 3) To develop new image processing tools which leverage prior biophysical knowledge to improve the reconstruction and quantification of Golgi morphology. These three methodological developments will be applied to a novel synthetic biology system that 'landlocks' Golgi cisternae to mitochondria and will facilitate favorable geometries to monito Golgi function. Although targeted at the Golgi, our methodological developments will be broadly applicable to live-cell super-resolution dynamic imaging of nearly every organelle within the cell.

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  • 1.Platelet P-selectin initiates cross-presentation and dendritic cell differentiation in blood monocytes by Han et al. (vol 6, eaaz1580, 2020)

    • 关键词：
    • IMMUNOLOGICAL SYNAPSE; GLYCOPROTEIN LIGAND-1; INTEGRIN ACTIVATION;HIGH-AVIDITY; T-CELLS; ANTIGEN; IMMUNE; INDUCTION; RECEPTOR; KINASE

    Dendritic cells (DCs) are adept at cross-presentation and initiation of antigen-specific immunity. Clinically, however, DCs produced by in vitro differentiation of monocytes in the presence of exogenous cytokines have been met with limited success. We hypothesized that DCs produced in a physiological manner may be more effective and found that platelets activate a cross-presentation program in peripheral blood monocytes with rapid (18 hours) maturation into physiological DCs (phDCs). Differentiation of monocytes into phDCs was concomitant with the formation of an "adhesion synapse," a biophysical junction enriched with platelet P-selectin and monocyte P-selectin glycoprotein ligand 1, followed by intracellular calcium fluxing and nuclear localization of nuclear factor.B. phDCs were more efficient than cytokine-derived DCs in generating tumor-specific T cell immunity. Our findings demonstrate that platelets mediate a cytokine-independent, physiologic maturation of DC and suggest a novel strategy for DC-based immunotherapies.

    ...

  • 3.Acylation A New Means to Control Traffic Through the Golgi

    • 关键词：
    • Golgi; palmitoylation; acylation; anterograde transport; Golgi bypass;membrane traffic;GLUT4 GLUCOSE TRANSPORTERS; ACYL-COENZYME-A; PROTEIN PALMITOYLATION;INSULIN ACTION; SECRETORY CARGO; MEMBRANE CARGO; DOMAIN; TRANSLOCATION;MECHANISMS; COPI

    The Golgi is well known to act as center for modification and sorting of proteins for secretion and delivery to other organelles. A key sorting step occurs at the trans-Golgi network and is mediated by protein adapters. However, recent data indicate that sorting also occurs much earlier, at the cis-Golgi, and uses lipid acylation as a novel means to regulate anterograde flux. Here, we examine an emerging role of S-palmitoylation/acylation as a mechanism to regulate anterograde routing. We discuss the critical Golgi-localized DHHC S-palmitoyltransferase enzymes that orchestrate this lipid modification, as well as their diverse protein clients (e.g., MAP6, SNAP25, CSP, LAT, beta-adrenergic receptors, GABA receptors, and GLUT4 glucose transporters). Critically, for integral membrane proteins, S-acylation can act as new a "self-sorting" signal to concentrate these cargoes in rims of Golgi cisternae, and to promote their rapid traffic through the Golgi or, potentially, to bypass the Golgi. We discuss this mechanism and examine its potential relevance to human physiology and disease, including diabetes and neurodegenerative diseases.

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