Study of Protein Translocation Using Microfluidic Electroporative Flow Cytometry

利用微流控电穿孔流式细胞术研究蛋白质易位

基本信息

项目摘要

Technologies based on data from single cells within a large population, most notably flow cytometry, have been successfully applied to study biological problems associated with dynamics in the protein expression level. In comparison, the determination of protein translocation within cells (i.e. movement of a protein between the cytoplasm and plasma membrane, or between the nucleus and cytoplasm) has been only carried out by bulk sample methods such as fractionation/Western blotting or imaging of a low number of cells. Protein translocation has fundamental importance in biology and medicine. Although the vast majority of proteins are synthesized in the cytoplasm, 20% proteins are located in non-cytoplasmic aqueous spaces and additional 25-30% of the proteins are located within a membrane. Studying protein translocation is critical for understanding signal transduction and regulation pathways in cells and the disease processes that they are involved in. Translocation does not involve change in the protein expression level therefore is undetectable by conventional flow cytometry. Thus high throughput techniques for detecting protein translocation in single cells are in great demand for generating mechanistic insights into a lot of important biological processes. In this project, our overall goal is to develop a new high throughput technique, which we refer to as microfluidic electroporative flow cytometry (EFC), to detect the protein translocation at the single cell level and study the kinetics of translocation processes. Microfluidic EFC combines electroporation (the application of an external electrical field to breach the cell membrane barrier) with flow cytometry. Our preliminary data indicate that the release of a protein from cells into surrounding solution during electroporation is dependent on its subcellular location. By recording the loss in the protein amount due to electroporation for a single cell, we will be able to determine whether translocation occurs. Using this approach we will test two model proteins that are biologically important: a kinase Syk which translocates from the cytoplasm to the plasma membrane and a transcription factor NF-kappaB which transports from the cytoplasm to the nucleus upon stimulation. We will demonstrate detection of these two different types of translocations at the single cell level. Furthermore we will use microfluidic EFC to generate data on the kinetics of these processes under different stimulation conditions. We will provide cross-platform validation of the technology by comparing the results to those obtained using traditional methods such as fractionation/western blotting and confocal fluorescence microscopy. As the integrated educational activities, we will train undergraduate and graduate students in interdisciplinary settings with emphasis on women and underrepresented minority students and disseminate the knowledge to high school students and the general public.
基于大群体中单细胞数据的技术,尤其是流式细胞术,已经成功地应用于研究与蛋白质表达水平动态相关的生物学问题。相比之下,细胞内蛋白质易位的测定(即蛋白质在细胞质和质膜之间或细胞核和细胞质之间的运动)只能通过大量样品方法进行,如分离/Western blotting或少量细胞的成像。蛋白质易位在生物学和医学中具有重要的基础意义。虽然绝大多数蛋白质是在细胞质中合成的,但20%的蛋白质位于非细胞质的水空间中,另外25-30%的蛋白质位于膜内。研究蛋白质易位对于理解细胞中的信号转导和调控途径以及它们所参与的疾病过程至关重要。易位不涉及蛋白质表达水平的改变,因此传统流式细胞术无法检测到。因此,检测单细胞中蛋白质易位的高通量技术对于产生对许多重要生物过程的机制见解有很大的需求。在本项目中,我们的总体目标是开发一种新的高通量技术,我们称之为微流体电穿孔流式细胞术(EFC),以检测单细胞水平的蛋白质易位并研究易位过程的动力学。微流控EFC结合了电穿孔(应用外电场突破细胞膜屏障)和流式细胞术。我们的初步数据表明,在电穿孔过程中,蛋白质从细胞释放到周围溶液中取决于其亚细胞位置。通过记录单个细胞因电穿孔导致的蛋白质量损失,我们将能够确定是否发生易位。使用这种方法,我们将测试两种具有重要生物学意义的模型蛋白:从细胞质转运到质膜的激酶Syk和在刺激下从细胞质转运到细胞核的转录因子NF-kappaB。我们将展示在单细胞水平上检测这两种不同类型的易位。此外,我们将使用微流控EFC来生成这些过程在不同刺激条件下的动力学数据。我们将通过将结果与传统方法(如分离/western blotting和共聚焦荧光显微镜)获得的结果进行比较,提供该技术的跨平台验证。作为综合教育活动,我们将在跨学科的背景下培养本科生和研究生,重点是妇女和少数民族学生,并向高中学生和公众传播知识。

项目成果

期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)

数据更新时间:{{ journalArticles.updateTime }}

{{ item.title }}
{{ item.translation_title }}
  • DOI:
    {{ item.doi }}
  • 发表时间:
    {{ item.publish_year }}
  • 期刊:
  • 影响因子:
    {{ item.factor }}
  • 作者:
    {{ item.authors }}
  • 通讯作者:
    {{ item.author }}

数据更新时间:{{ journalArticles.updateTime }}

{{ item.title }}
  • 作者:
    {{ item.author }}

数据更新时间:{{ monograph.updateTime }}

{{ item.title }}
  • 作者:
    {{ item.author }}

数据更新时间:{{ sciAawards.updateTime }}

{{ item.title }}
  • 作者:
    {{ item.author }}

数据更新时间:{{ conferencePapers.updateTime }}

{{ item.title }}
  • 作者:
    {{ item.author }}

数据更新时间:{{ patent.updateTime }}

Chang Lu其他文献

Efficiently answering top-k frequent term queries in temporal-categorical range
有效回答时间分类范围内的前 k 个频繁术语查询
  • DOI:
    10.1016/j.ins.2021.05.081
  • 发表时间:
    2021
  • 期刊:
  • 影响因子:
    8.1
  • 作者:
    Zhenying He;Lu Wang;Chang Lu;Yinan Jing;Kai Zhang;Weili Han;Jianxin Li;Chengfei Liu;Xiaoyang Sean Wang
  • 通讯作者:
    Xiaoyang Sean Wang
Vertically aligned sheet-like structural aerogel incorporated with expanded graphite for efficient solar desalination and atmospheric water harvesting
垂直排列的片状结构气凝胶与膨胀石墨相结合,可实现高效的太阳能海水淡化和大气集水
  • DOI:
    10.1016/j.cej.2024.153470
  • 发表时间:
    2024
  • 期刊:
  • 影响因子:
    15.1
  • 作者:
    Ning Ding;Bo Liang;Xi;Dahu Yao;Jing Chen;Cuiyun Liu;Chang Lu;Xinchang Pang
  • 通讯作者:
    Xinchang Pang
Explosion overpressure behavior and flame propagation characteristics in hybrid explosions of hydrogen and magnesium dust
氢镁粉尘混合爆炸中爆炸超压行为及火焰传播特性
  • DOI:
    10.1016/j.fuel.2022.125801
  • 发表时间:
    2022
  • 期刊:
  • 影响因子:
    7.4
  • 作者:
    Wentao Ji;Yang Wang;Jingjing Yang;Jia He;Chang Lu;Xiaoping Wen;Yan Wang
  • 通讯作者:
    Yan Wang
DNA‐directed Seeded Synthesis of Gold Nanoparticles without Changing DNA Sequence
不改变 DNA 序列的 DNA 定向种子合成金纳米颗粒
  • DOI:
    10.1002/cnma.202200111
  • 发表时间:
    2022-05
  • 期刊:
  • 影响因子:
    3.8
  • 作者:
    Chang Lu;Mohamad Z;ieh;Jingkai Zheng;Juewen Liu
  • 通讯作者:
    Juewen Liu
Pharmacological mechanism of Shenlingbaizhu formula against experimental colitis
参苓白术方抗实验性结肠炎的药理机制
  • DOI:
    10.1016/j.phymed.2022.153961
  • 发表时间:
  • 期刊:
  • 影响因子:
    7.9
  • 作者:
    Jing Yan;Wei Yu;Chang Lu
  • 通讯作者:
    Chang Lu

Chang Lu的其他文献

{{ item.title }}
{{ item.translation_title }}
  • DOI:
    {{ item.doi }}
  • 发表时间:
    {{ item.publish_year }}
  • 期刊:
  • 影响因子:
    {{ item.factor }}
  • 作者:
    {{ item.authors }}
  • 通讯作者:
    {{ item.author }}

{{ truncateString('Chang Lu', 18)}}的其他基金

CAREER:Transfected cell microarray technology based on microfluidic electroporation
职业:基于微流控电穿孔的转染细胞微阵列技术
  • 批准号:
    1016547
  • 财政年份:
    2010
  • 资助金额:
    $ 34万
  • 项目类别:
    Continuing Grant
CAREER:Transfected cell microarray technology based on microfluidic electroporation
职业:基于微流控电穿孔的转染细胞微阵列技术
  • 批准号:
    0747105
  • 财政年份:
    2008
  • 资助金额:
    $ 34万
  • 项目类别:
    Continuing Grant

相似国自然基金

有翅与无翅蚜虫差异分泌唾液蛋白Cuticular protein在调控植物细胞壁免疫中的功能
  • 批准号:
    32372636
  • 批准年份:
    2023
  • 资助金额:
    50.00 万元
  • 项目类别:
    面上项目
抑制Protein Kinase D促进胚胎干细胞自我更新的分子机制研究
  • 批准号:
  • 批准年份:
    2022
  • 资助金额:
    54 万元
  • 项目类别:
    面上项目
C2 DOMAIN PROTEIN 1 (C2DP1)基因家族在植物开花调控中的功能研究
  • 批准号:
    n/a
  • 批准年份:
    2022
  • 资助金额:
    0.0 万元
  • 项目类别:
    省市级项目
凡纳滨对虾Laccase-like protein非酶活依赖参与抗WSSV免疫的分子机制
  • 批准号:
    n/a
  • 批准年份:
    2022
  • 资助金额:
    10.0 万元
  • 项目类别:
    省市级项目
帽结合蛋白(cap binding protein)调控乙烯信号转导的分子机制
  • 批准号:
    32170319
  • 批准年份:
    2021
  • 资助金额:
    58.00 万元
  • 项目类别:
    面上项目
帽结合蛋白(cap binding protein)调控乙烯信号转导的分子机制
  • 批准号:
  • 批准年份:
    2021
  • 资助金额:
    58 万元
  • 项目类别:
玉米基因Dirigent protein 4的克隆和功能鉴定
  • 批准号:
  • 批准年份:
    2021
  • 资助金额:
    30 万元
  • 项目类别:
    青年科学基金项目
C1q/TNF-related protein 9调控平滑肌细胞程序性坏死抑制动脉粥样硬化的机制研究
  • 批准号:
    81900309
  • 批准年份:
    2019
  • 资助金额:
    21.0 万元
  • 项目类别:
    青年科学基金项目

相似海外基金

Controlled Protein Translocation in Nanopores for Sequencing Applications
用于测序应用的纳米孔中的受控蛋白质易位
  • 批准号:
    10645979
  • 财政年份:
    2023
  • 资助金额:
    $ 34万
  • 项目类别:
Controlled Protein Translocation in Nanopores for Sequencing Applications
用于测序应用的纳米孔中的受控蛋白质易位
  • 批准号:
    10574679
  • 财政年份:
    2023
  • 资助金额:
    $ 34万
  • 项目类别:
Cancer progression-related translocation of gap junction protein into the Golgi apparatus – Elucidation of its molecular mechanism
癌症进展相关的间隙连接蛋白易位至高尔基体
  • 批准号:
    22K06996
  • 财政年份:
    2022
  • 资助金额:
    $ 34万
  • 项目类别:
    Grant-in-Aid for Scientific Research (C)
Molecular mechanisms of bacterial type III protein translocation across membranes
细菌III型蛋白跨膜易位的分子机制
  • 批准号:
    RGPIN-2018-05999
  • 财政年份:
    2022
  • 资助金额:
    $ 34万
  • 项目类别:
    Discovery Grants Program - Individual
Exploring protein translocation by the Legionella pneumophila Dot/Icm Type IV Section System
探索嗜肺军团菌 Dot/Icm IV 型切片系统的蛋白质易位
  • 批准号:
    10426349
  • 财政年份:
    2021
  • 资助金额:
    $ 34万
  • 项目类别:
Exploring protein translocation by the Legionella pneumophila Dot/Icm Type IV Section System
探索嗜肺军团菌 Dot/Icm IV 型切片系统的蛋白质易位
  • 批准号:
    10314686
  • 财政年份:
    2021
  • 资助金额:
    $ 34万
  • 项目类别:
Measurement of protein translocation by SecA ATPase
通过 SecA ATPase 测量蛋白质易位
  • 批准号:
    21K19226
  • 财政年份:
    2021
  • 资助金额:
    $ 34万
  • 项目类别:
    Grant-in-Aid for Challenging Research (Exploratory)
Molecular mechanisms of bacterial type III protein translocation across membranes
细菌III型蛋白跨膜易位的分子机制
  • 批准号:
    RGPIN-2018-05999
  • 财政年份:
    2021
  • 资助金额:
    $ 34万
  • 项目类别:
    Discovery Grants Program - Individual
Dynamic Structural Analysis of Protein Translocation Across Membrane by Cryo-EM
利用冷冻电镜对蛋白质跨膜易位进行动态结构分析
  • 批准号:
    21H02452
  • 财政年份:
    2021
  • 资助金额:
    $ 34万
  • 项目类别:
    Grant-in-Aid for Scientific Research (B)
Defining the mechanism of protein translocation using a bacterial exotoxin as a model system
使用细菌外毒素作为模型系统定义蛋白质易位机制
  • 批准号:
    10447290
  • 财政年份:
    2020
  • 资助金额:
    $ 34万
  • 项目类别:
{{ showInfoDetail.title }}

作者:{{ showInfoDetail.author }}

知道了