Ohse 赠款基金会资助了这项工作。

1. 细胞扩展特性
<ol><li>在标准的37.0 摄氏度和 5% CO2中, 用10% 胎牛血清和 1万 U/毫升青霉素和链霉素 (指定为 "生长培养基"), 将 SW-13、SW 和 SW-DKK3 细胞维持在标准化的湿润孵化箱中, 并在 Dulbecco 的改良后的鹰培养基中补充。<ol>
<li>使用 hemocytometer 计数细胞, 并将5000个细胞 SW-13, SW-新, SW-DKK3 线分成单独的6井板, 在无菌玻璃盖玻片的生长培养基中一夜之间生长。</li>
	</ol>
</li>
	<li>在一夜之间生长, 为每井, 吸入生长培养基, 洗涤细胞与1毫升温暖磷酸盐缓冲盐水 (PBS), 然后修复细胞与1毫升3.7% 甲醛为10分钟。</li>
	<li>吸入甲醛和染色细胞在每个井与1毫升0.05% 水晶紫30分钟. 用三洗涤1毫升去离子水清洗过量染料。 注意: 根据染料摄取的水平, 可能需要额外的一些洗涤来去除背景染色, 以促进细胞的可视化。<ol>
<li>使用细尖钳, 检索盖玻片, 并把他们脸上的标签玻璃幻灯片与一滴清晰的安装试剂。</li>
	</ol>
</li>
	<li>在光镜下, 随机选择20个不同的细胞, 从每个盖玻片的细胞延伸试验。<ol>
<li>以每个视野的400x 放大倍数显微照片。直接计算20个代表单元格中每个扩展类型的数目。 注: 因此, 应检查每一个测试的条件, 20 个独立的细胞, 以确保可靠的结果。丝状伪足是由细胞扩展定义的, 其基部为5微米或更小, 单顶点。Lobopodia 是由细胞扩展定义的, 其基数为5到20微米, 包含有多个顶端的扩展。Lamellipodia 的定义是细胞扩展, 长度大于20微米, 有或没有顶端。根据已测试的单元格类型, 单元格扩展的大小可能会有所不同, 并且可能需要对识别参数进行细化。</li>
	</ol>
</li>
	<li>确定每个单元格类型中每个单元格扩展的平均数目 (即、SW-13、sw-DDK), 并检查了6个水井。</li>
	<li>采用单向方差分析 (方差) 统计试验, 比较不同试验细胞类型间细胞膜延长平均百分率的差异。</li>
</ol>2. 黏附力试验
<ol><li>在37.0 摄氏度和 5% CO2的生长培养基中, 维持 SW-13、SW 和 SW-DKK3 细胞。<ol>
<li>使用 hemocytometer, 计数和板10万细胞的 SW-13, SW-新, 和 SW-DKK3 每井成单独的6井板和生长在一夜之间生长培养基。</li>
		<li>种子6井板为每个细胞类型测试, 使用一个板材为每6指定的时间点测试下面。 注意: 不同的单元格类型的时间点可能会有所不同。</li>
	</ol>
</li>
	<li>夜间孵化后, 用温暖的 PBS 冲洗细胞, 然后加入0.5 毫升的1x 非酶细胞离解溶液, 每一个井。<ol>
<li>旋转板块, 传播细胞离解溶液。在已定义的时间点 (即1、2、3、5、10和15分钟) 中吸入指定的板, 然后用1毫升温 PBS 解决方案三次清洗。</li>
		<li>在洗涤之间, 轻轻地敲击盘子以分离松散附着的细胞。</li>
	</ol>
</li>
	<li>吸入任何剩余的 PBS, 并固定在板上剩余的细胞与1毫升3.7% 甲醛10分钟。</li>
	<li>染色细胞与1毫升0.05% 晶体紫30分钟然后用1毫升的去离子水洗涤多余的染料。滴定洗涤促进细胞可视化。 注意: 根据染料摄取的水平, 可能需要额外的一些洗涤来促进细胞的可视化。</li>
	<li>使用光显微镜在100x 放大倍数, 计数剩余的连接的细胞在每个井为每个板材。 注意: 使用透明标尺或在板的底部标记细笔参考线将有助于避免重复计数相同的单元格。</li>
	<li>确定每个板块每个井的平均附着单元数。</li>
	<li>采用双向方差分析统计试验, 比较 SW-13、SW、SW-DDK3 细胞在给定时间段内的细胞脱离率。</li>
</ol>

<ol><li>Libe, R., Fratticci, A., Bertherat, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adrenocortical+cancer%3A+pathophysiology+and+clinical+management%5BTitle%5D)+AND+%22Endocrine-related+cancer%22%5BJournal%5D)">Adrenocortical cancer: pathophysiology and clinical management.</a> Endocrine-related cancer. 14 (1), 13-28 (2007).</li>
<li>Veeck, J., Dahl, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeting+the+Wnt+pathway+in+cancer%3A+the+emerging+role+of+Dickkopf-3%5BTitle%5D)+AND+%22Biochimica+et+biophysica+acta%22%5BJournal%5D)">Targeting the Wnt pathway in cancer: the emerging role of Dickkopf-3.</a> Biochimica et biophysica acta. 1825 (1), 18-28 (2012).</li>
<li>Friedl, P., Gilmour, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Collective+cell+migration+in+morphogenesis%2C+regeneration+and+cancer%5BTitle%5D)+AND+%22Nat+Rev+Mol+Cell+Biol%22%5BJournal%5D)">Collective cell migration in morphogenesis, regeneration and cancer.</a> Nat Rev Mol Cell Biol. 10 (7), 445-457 (2009).</li>
<li>Jacquemet, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((L-type+calcium+channels+regulate+filopodia+stability+and+cancer+cell+invasion+downstream+of+integrin+signalling%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">L-type calcium channels regulate filopodia stability and cancer cell invasion downstream of integrin signalling.</a> Nat Commun. 7, 13297(2016).</li>
<li>Machesky, L. M., Li, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fascin%3A+Invasive+filopodia+promoting+metastasis%5BTitle%5D)+AND+%22Commun+Integr+Biol%22%5BJournal%5D)">Fascin: Invasive filopodia promoting metastasis.</a> Commun Integr Biol. 3 (3), 263-270 (2010).</li>
<li>Petrie, R. J., Harlin, H. M., Korsak, L. I., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Activating+the+nuclear+piston+mechanism+of+3D+migration+in+tumor+cells%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Activating the nuclear piston mechanism of 3D migration in tumor cells.</a> J Cell Biol. 216 (1), 93-100 (2017).</li>
<li>Korah, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epigenetic+silencing+of+RASSF1A+deregulates+cytoskeleton+and+promotes+malignant+behavior+of+adrenocortical+carcinoma%5BTitle%5D)+AND+%22Molecular+cancer%22%5BJournal%5D)">Epigenetic silencing of RASSF1A deregulates cytoskeleton and promotes malignant behavior of adrenocortical carcinoma.</a> Molecular cancer. 12, 87(2013).</li>
<li>Cheng, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+FOXO1-mediated+dedifferentiation+blocking+role+for+DKK3+in+adrenocortical+carcinogenesis%5BTitle%5D)+AND+%22BMC+cancer%22%5BJournal%5D)">A novel FOXO1-mediated dedifferentiation blocking role for DKK3 in adrenocortical carcinogenesis.</a> BMC cancer. 17 (1), 164(2017).</li>
<li>Barry, D. J., Durkin, C. H., Abella, J. V., Way, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Open+source+software+for+quantification+of+cell+migration%2C+protrusions%2C+and+fluorescence+intensities%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Open source software for quantification of cell migration, protrusions, and fluorescence intensities.</a> J Cell Biol. 209 (1), 163-180 (2015).</li>
<li>Cliffe, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+3D+analysis+of+complex+single+border+cell+behaviors+in+coordinated+collective+cell+migration%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Quantitative 3D analysis of complex single border cell behaviors in coordinated collective cell migration.</a> Nat Commun. 8, 14905(2017).</li>
</ol>

作者没有什么可透露的。

在这里, 我们描述了一个体外定量的方法来表征细胞扩展的简便, 少坑瀑布, 可靠的重现性, 可用于各种测试条件。此外, 简单, 可量化的黏附力和/或运动分析可以同时执行, 以关联潜在的功能意义的观察改变的细胞膜延长。
然而, 这些方法可能会带来一些潜在的限制。首先, 这里指定的标准, 以确定不同类型的细胞扩展是基于肾上腺皮质 SW-13 细胞形态学, 因此可能需要细?...

失调 wnt 信号在肾上腺皮质恶性肿瘤中起重要作用1。本研究中使用的方法研究了 DKK3 的沉默, 即 WNT 信号的负调节因子, 是否代表肾上腺皮质的分化事件, 并在细胞扩展曲目变化的背景下促进肿瘤的形成。DKK3 是一个 38 kDa 分泌糖蛋白与 n-终端信号肽和以往的研究表明, 其强迫表达导致细胞周期骤停, 抑制侵袭性恶性行为, 并逆转上皮间充质转换2。
肾上腺皮质癌 (ACC) 和其他癌症的恶性行为在某种程度上受肿瘤细胞与周围表面相互作用的能力的影响, 包括细胞外基质, 进而促进肿瘤细胞侵袭和迁移3。特定细胞膜延伸在癌症进展中的作用正越来越多地表现在不同的语境中, 主要是通过丝状伪足的形成。例如, L 型钙通道的过度表达诱导丝状伪足形成, 促进肿瘤细胞侵袭 4.同样, 在正常组织中最小表达的肌动蛋白结合蛋白 Fascin 也抗原与丝状伪足形成5相关联的癌细胞.Lobopodia 的形成使非恶性成纤维细胞能够有效地通过超细胞基质迁移, 然而, 它已经表明, 肉瘤细胞依靠金属蛋白酶的活动代替 Lobopodia, 以促进细胞迁移和入侵6。我们已经表明, 肿瘤抑制器, 包括 Ras 协会域家族1异构体 A (RASSF1A) 和 DKK3, 可以发挥作用, 以改变骨架元素和促进 lamellipodia 形成和阻碍侵入性属性7,8。
因此, 重要的是要描述发生癌变的基因的影响及其与细胞膜延长改变的关系, 特别是评估丝状伪足、lobopodia 和 lamellipodia 在试验条件下的形成。目前的先进技术包括使用越来越复杂的显微方法、荧光标记和/或复杂的计算机算法来获取和解释数据。虽然这些方法提供了新的强大的分析工具, 但它们的复杂性限制了它们在细胞生物学实验中的广泛应用和适应性。此外, 对单元格扩展形态学变化的精确量化和观察通常不被测量9,10。相比之下, 我们在这里引入了一种技术, 通过标准显微技术和易于适应的体外方法, 精确地量化细胞扩展的变化。这些方法还量化每个细胞的扩展类型同时为每个细胞分析和确定的整体变化的细胞膜延长曲目。我们还展示了这些变化如何与细胞黏附性质有关。
作为一个实验性的例子, 我们将使用以前创建的 SW-13 细胞系, 指定的 SW-DDK3, 它已经稳定地转染了 pCMV6-Entry/DKK3 质粒载体和组成性 overexpresses DKK3, 一个区别因素在肾上腺。非转染的 SW-13 细胞和 SW-13 细胞稳定转染的空载体 (pCMV6-Entry), 指定的 SW, 将作为实验控制。

<table><tbody><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>ThermoFisher</td><td>11965-092</td><td>Supplemented with fetal bovine serum and penicllin and streptomycin</td></tr><tr><td>Deulbeco&#039;s Phosphate Buffered Saline</td><td>Sigma-Aldrich</td><td>D8537</td><td>1X solution, used directly</td></tr><tr><td>3.7% formaldehyde&amp;nbsp;</td><td>Sigma-Aldrich</td><td>F8775</td><td>Dilute stock solution</td></tr><tr><td>0.05% crystal violet&amp;nbsp;</td><td>Harleco</td><td>192-12</td><td>Dilute stock solution</td></tr><tr><td>De-ionized water</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td>Microscope with 400X magnification&amp;nbsp;</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td>6-well plates</td><td>Corning</td><td>353046</td><td>NA</td></tr><tr><td>Cell dissociation solution non-enzymatic 1X</td><td>Sigma-Aldrich</td><td>C5914&amp;nbsp;</td><td>1X solution, used directly</td></tr></tbody></table>

characterization of cell membrane extensions and studying their roles in cancer cell adhesion dynamics

细胞膜的扩展曲目调节癌细胞的各种恶性行为, 包括它们的粘合剂和迁移电位。准确分类和量化细胞扩展的能力和测量细胞的粘附能力对确定细胞信号事件如何影响癌细胞的行为和侵略性至关重要。在这里, 我们描述了体外的设计和使用的细胞扩展量化方法结合的黏附能力检测在建立的体外模型的肾上腺皮质癌 (ACC)。具体来说, 我们测试 DKK3, 一个假定的肿瘤抑制物和赞成分化因子, 对 ACC 细胞系, SW-13 的膜扩张表型的影响。我们建议这些化验提供相对简单, 可靠, 容易解释的指标, 以衡量这些特点在各种实验条件下。

采用上述检测方法, 对建立的 ACC 细胞系 SW-13、体外进行了 DKK3 过度表达对细胞增殖形态学和细胞黏附性能的影响。细胞 overexpressing DKK3 是由稳定的转染 SW-13 细胞与 c-myc DDK 标记 pCMV6-Entry/DKK3 质粒载体, 并指定为 SW-DKK3。同样地, 以空矢量 pCMV6-Entry 稳定转染的细胞也被创建为传代控制, 并被定义为 SW-尼奥。SW-13 细胞被用作额外的父细胞控制。定量实时 PCR 和西方印迹技...

Watch this Scientific Journal Video about Characterization of Cell Membrane Extensions and Studying Their Roles in Cancer Cell Adhesion Dynamics at JoVE.com

Characterization of Cell Membrane Extensions and Studying Their Roles in Cancer Cell Adhesion Dynamics

本研究表明定量细胞膜延伸测量的实用性和易用性, 及其与细胞粘附能力的相关性。作为一个代表性的例子, 我们在这里表明, Dickkopf 相关蛋白 3 (DKK3) 促进增加 lobopodia 形成和细胞粘附在肾上腺皮质癌细胞在体外。

细胞膜延伸特性及其在癌细胞黏附动力学中的作用研究

The cell membrane&#39;s extension repertoire modulates various malignant behaviors of cancer cells, including their adhesive and migratory potentials. The ...

characterization-cell-membrane-extensions-studying-their-roles-cancer

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

8.3K 次观看.  Yale University School of Medicine. 本研究表明定量细胞膜延伸测量的实用性和易用性, 及其与细胞粘附能力的相关性。作为一个代表性的例子, 我们在这里表明, Dickkopf 相关蛋白 3 (DKK3) 促进增加 lobopodia 形成和细胞粘附在肾上腺皮质癌细胞在体外。

视频: Characterization of Cell Membrane Extensions and Studying Their Roles in Cancer Cell Adhesion Dynamics

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

3维建模和成像集体细胞的侵袭

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

科研

医学

3-D Cell Culture System for Studying Invasion and Evaluating Therapeutics in Bladder Cancer

Bladder cancer is a significant health problem. It is estimated that more than 16,000 people will die this year in the United States from bladder cancer. While 75% of bladder cancers are non-invasive and unlikely to metastasize, about 25% progress to an invasive growth pattern. Up to half of the patients with invasive cancers will develop lethal metastatic relapse. Thus, understanding the mechanism of invasive progression in bladder cancer is crucial to predict patient outcomes and prevent lethal metastases. In this article, we present a three-dimensional cancer invasion model which allows incorporation of tumor cells and stromal components to mimic in vivo conditions occurring in the bladder tumor microenvironment. This model provides the opportunity to observe the invasive process in real time using time-lapse imaging, interrogate the molecular pathways involved using confocal immunofluorescent imaging and screen compounds with the potential to block invasion. While this protocol focuses on bladder cancer, it is likely that similar methods could be used to examine invasion and motility in other tumor types as well.

The processes governing bladder cancer invasion represent opportunities for biomarker and therapeutic development. Here we present a bladder cancer invasion model which incorporates 3-D culture of tumor spheroids, time-lapse imaging and confocal microscopy. This technique is useful for defining the features of the invasive process and for screening therapeutic agents.

3维细胞培养系统研究膀胱癌侵袭和评价治疗方法

Bladder cancer is a significant health problem. It is estimated that more than 16,000 people will die this year in the United States from bladder cancer. ...

癌症研究

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

在一个三维胶原蛋白矩阵分析细胞迁移

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

生物工程

Examining the Dynamics of Cellular Adhesion and Spreading of Epithelial Cells on Fibronectin During Oxidative Stress

The adhesion and spreading of cells onto the extracellular matrix (ECM) are essential cellular processes during organismal development and for the homeostasis of adult tissues. Interestingly, oxidative stress can alter these processes, thus contributing to the pathophysiology of diseases such as metastatic cancer. Therefore, understanding the mechanism(s) of how cells attach and spread on the ECM during perturbations in redox status can provide insight into normal and disease states. Described below is a step-wise protocol that utilizes an immunofluorescence-based assay to specifically quantify cell adhesion and spreading of immortalized fibroblast cells on fibronectin (FN) in vitro. Briefly, anchorage-dependent cells are held in suspension and exposed to the ATM kinase inhibitor Ku55933 to induce oxidative stress. Cells are then plated on FN-coated surface and allowed to attach for predetermined periods of time. Cells that remain attached are fixed and labeled with fluorescence-based antibody markers of adhesion (e.g., paxillin) and spreading (e.g., F-actin). Data acquisition and analysis are performed using commonly available laboratory equipment, including an epifluorescence microscope and freely available Fiji software. This procedure is highly versatile and can be modified for a variety of cell lines, ECM proteins, or inhibitors in order to examine a broad range of biological questions.

This method is useful for quantifying the early dynamics of cellular adhesion and spreading of anchorage-dependent cells onto the fibronectin. Furthermore, this assay can be used to investigate the effects of altered redox homeostasis on cell spreading and/or cell adhesion-related intracellular signaling pathways.

在氧化应激期间细胞粘附和纤维蛋白扩散动力学

The adhesion and spreading of cells onto the extracellular matrix (ECM) are essential cellular processes during organismal development and for the ...

生物化学

Revealing the Cytoskeletal Organization of Invasive Cancer Cells in 3D

Cell migration has traditionally been studied in 2D substrates. However, it has become increasingly evident that there is a need to study cell migration in more appropriate 3D environments, which better resemble the dimensionality of the physiological processes in question. Migratory cells can substantially differ in their morphology and mode of migration depending on whether they are moving on 2D or 3D substrates. Due to technical difficulties and incompatibilities with most standard protocols, structural and functional analysis of cells embedded within 3D matrices still remains uncommon. This article describes methods for preparation and imaging of 3D cancer cell cultures, either as single cells or spheroids. As an appropriate ECM substrate for cancer cell migration, we use nonpepsinized rat tail collagen I polymerized at room-temperature and fluorescently labeled to facilitate visualization using standard confocal microscopes. This work also includes a protocol for 3D immunofluorescent labeling of endogenous cell cytoskeleton. Using these protocols we hope to contribute to a better description of the molecular composition, localization, and functions of cellular structures in 3D.

This article presents a method to fluorescently label collagen that can be further used for both fix and live imaging of 3D cell cultures. We also provide an optimized protocol to visualize endogenous cytoskeletal proteins of cells cultured in 3D environments.

在3D揭示侵入性癌症细胞骨架组织

Cell migration has traditionally been studied in 2D substrates. However, it has become increasingly evident that there is a need to study cell migration ...

Nanopodia - Thin, Fragile Membrane Projections with Roles in Cell Movement and Intercellular Interactions

Adherent cells in culture maintain a polarized state to support movement and intercellular interactions. Nanopodia are thin, elongated, largely F-actin-negative membrane projections in endothelial and cancer cells that can be visualized through TM4SF1 (Transmembrane-4-L-six-family-1) immunofluorescence staining. TM4SF1 clusters in 100-300 &#956;m diameter TMED (TM4SF1 enriched microdomains) containing 3 to as many as 14 individual TM4SF1 molecules. TMED are arranged intermittently along nanopodia at a regular spacing of 1 to 3 TMED per &#956;m and firmly anchor nanopodia to matrix. This enables nanopodia to extend more than 100 &#956;m from the leading front or trailing rear of polarized endothelial or tumor cells, and causes membrane residues to be left behind on matrix when the cell moves away. TMED and nanopodia have been overlooked because of their extreme fragility and sensitivity to temperature. Routine washing and fixation disrupt the structure. Nanopodia are preserved by direct fixation in paraformaldehyde (PFA) at 37 &#176;C, followed by brief exposure to 0.01% Triton X-100 before staining. Nanopodia open new vistas in cell biology: they promise to reshape our understanding of how cells sense their environment, detect and identify other cells at a distance, initiate intercellular interactions at close contact, and of the signaling mechanisms involved in movement, proliferation, and cell-cell communications. The methods that are developed for studying TM4SF1-derived nanopodia may be useful for studies of nanopodia that form in other cell types through the agency of classic tetraspanins, notably the ubiquitously expressed CD9, CD81, and CD151.

Nanopodia are thin but fragile membrane channels that extend up to 100 &#956;m from a cell's leading front or trailing rear and sense the cellular environment. Direct fixation at 37 &#176;C, gentle washing, and avoidance of organic solvents like ethanol, methanol, or acetone and of higher Triton X-100 concentrations are required to observe these cellular structures.

Nanopodia  - 薄，易碎膜预测与细胞运动和细胞间的相互作用角色

Adherent cells in culture maintain a polarized state to support movement and intercellular interactions. Nanopodia are thin, elongated, largely ...

生物学

Quantitative Analysis of Cell Edge Dynamics during Cell Spreading

Cell spreading is a dynamic process in which a cell suspended in media attaches to a substrate and flattens itself from a rounded to a thin and spread-out shape. Following the cell-substrate attachment, the cell forms a thin sheet of lamellipodia emanating from the cell body. In the lamellipodia, globular actin (G-actin) monomers polymerize into a dense filamentous actin (F-actin) meshwork that pushes against the plasma membrane, thereby providing the mechanical forces required for the cell to spread. Notably, the molecular players that control the actin polymerization in lamellipodia are essential for many other cellular processes, such as cell migration and endocytosis. 
Since spreading cells form continuous lamellipodia that span the entire cell periphery and persistently expand outward, cell spreading assays have become an efficient tool to assess the kinetics of lamellipodial protrusions. Although several technical implementations of the cell spreading assay have been developed, a detailed description of the workflow, which would include both a step-by-step protocol and computational tools for data analysis, is currently lacking. Here, we describe the experimental procedures of the cell spreading assay and present an open-source tool for quantitative and unbiased analysis of cell edge dynamics during spreading. When combined with pharmacological manipulations and/or gene-silencing techniques, this protocol is amenable to a large-scale screen of molecular players regulating lamellipodial protrusions.

In this protocol, we present the experimental procedures of a cell spreading assay that is based on live-cell microscopy. We provide an open-source computational tool for the unbiased segmentation of fluorescently labeled cells and quantitative analysis of lamellipodia dynamics during cell spreading.

细胞扩散过程中细胞边缘动力学的定量分析

Cell spreading is a dynamic process in which a cell suspended in media attaches to a substrate and flattens itself from a rounded to a thin and spread-out ...

Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of inflammation. This phenomenon occurs in postcapillary venules, where blood flow pushes leukocytes in a rolling motion on the endothelial cells. Stable rolling requires a delicate balance between adhesion bond formation and their mechanically-driven dissociation, allowing the cell to remain attached to the surface while rolling in the direction of flow. Unlike other adhesion processes occurring in relatively static environments, rolling adhesion is highly dynamic as the rolling cells travel over thousands of microns at tens of microns per second. Consequently, conventional mechanobiology methods such as traction force microscopy are unsuitable for measuring the individual adhesion events and the associated molecular forces due to the short timescale and high sensitivity required. Here, we describe our latest implementation of the adhesion footprint assay to image the P-selectin: PSGL-1 interactions in rolling adhesion at the molecular level. This method utilizes irreversible DNA-based tension gauge tethers to produce a permanent history of molecular adhesion events in the form of fluorescence tracks. These tracks can be imaged in two ways: (1) stitching together thousands of diffraction-limited images to produce a large field of view, enabling the extraction of adhesion footprint of each rolling cell over thousands of microns in length, (2) performing DNA-PAINT to reconstruct super-resolution images of the fluorescence tracks within a small field of view. In this study, the adhesion footprint assay was used to study HL-60 cells rolling at different shear stresses. In doing so, we were able to image the spatial distribution of the P-selectin: PSGL-1 interaction and gain insight into their molecular forces through fluorescence intensity. Thus, this method provides the groundwork for the quantitative investigation of the various cell-surface interactions involved in rolling adhesion at the molecular level.

This protocol presents the experimental procedures to perform the adhesion footprint assay to image the adhesion events during fast cell rolling adhesion.

通过粘附足迹测定法对细胞滚动中的分子粘附进行成像

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of ...

Measuring Cell-Edge Protrusion Dynamics during Spreading using Live-Cell Microscopy

The development and homeostasis of multicellular organisms rely on coordinated regulation of cell migration. Cell migration is an essential event in the construction and regeneration of tissues, and is critical in embryonic development, immunological responses, and wound healing. Dysregulation of cell motility contributes to pathological disorders, such as chronic inflammation and cancer metastasis. Cell migration, tissue invasion, axon, and dendrite outgrowth all initiate with actin polymerization-mediated cell-edge protrusions. Here, we describe a simple, efficient, time-saving method for the imaging and quantitative analysis of cell-edge protrusion dynamics during spreading. This method measures discrete features of cell-edge membrane dynamics, such as protrusions, retractions, and ruffles, and can be used to assess how manipulations of key actin regulators impact cell-edge protrusions in diverse contexts.

This protocol aims to measure the dynamic parameters (protrusions, retractions, ruffles) of protrusions at the edge of spreading cells.

使用活细胞显微镜测量扩散过程中的细胞边缘突起动力学

The development and homeostasis of multicellular organisms rely on coordinated regulation of cell migration. Cell migration is an essential event in the ...

Cell Adhesion Molecules - Types and Functions

Cell adhesion molecules (CAMs) are pivotal to multicellularity and the coordinated functioning of tissues and organ systems. They enable physical interactions between cells and provide mechanical strength to tissues. They also function as receptors for signal transmission across the plasma membrane. The CAMs are broadly classified into four families - integrins, cadherins, selectins, and immunoglobulin-like CAMs (IgCAMs).
CAM Families
The Integrin family of proteins is primarily&#160; involved in a cell&#8217;s interaction with its surrounding matrix. However, some integrins can bind CAMs on another cell&#8217;s surface and participate in direct cell-cell interactions. For example, the integrins on immune cells bind IgCAMs expressed on the vascular endothelium.
Cadherins are a superfamily of calcium-dependent glycoproteins primarily involved in establishing strong cell adhesions. They compose the adherens junctions and desmosomes in tissues, such as the epithelium.
Selectins and IgCAMs are involved in transient cell interactions and participate in directing cells towards target sites. For example, they help in the selective recruitment of lymphocytes to the secondary lymphoid organs.
CAM and Multicellularity
CAMs are found&#160; across virtually all multicellular organisms - from sponges and simple nematodes to complex invertebrates and vertebrates. The complexity of the cellular interactions, and therefore of the CAMs, increases with the complexity of the organisms. For example,&#160; while the fruit fly D.melanogaster has approximately&#160; 500 genes involved in cell adhesion, complex vertebrates like mammals have over a thousand genes that code for different types of CAMs.