The Ohse Grant Foundation funded this work.

1. Cell Extension Characteristics
<ol>
	<li>Maintain SW-13, SW-Neo, and SW-DKK3 cells in a standard humidified incubator at 37.0 &#176;C and 5% CO2 in Dulbecco&#39;s Modified Eagle Medium supplemented with 10% fetal bovine serum and 10,000 U/mL penicillin and streptomycin (designated as &#39;growth medium&#39;).
	<ol>
		<li>Count cells using a hemocytometer, and plate 5,000 cells per well for SW-13, SW-Neo, and SW-DKK3 lines into separate 6-well plates and grow overnight in growth medium on sterile glass coverslips.</li>
	</ol>
	</li>
	<li>After overnight growth, for each well, aspirate the growth medium, wash cells with 1 mL of warm phosphate-buffered saline (PBS), and then fix the cells with 1 mL of 3.7% formaldehyde for 10 min.</li>
	<li>Aspirate formaldehyde and stain cells in each well with 1 mL of 0.05% crystal violet for 30 min. Wash excess dye away using three washes of 1 mL deionized water. 
	NOTE: Depending on the level of dye uptake, several additional washes may be needed to remove background staining to promote cell visualization.
	<ol>
		<li>Using fine tipped forceps, retrieve coverslips and place them face down on a labeled glass slide with a drop of clear mounting reagent.</li>
	</ol>
	</li>
	<li>Under a light microscope, randomly choose 20 views of non-overlapping cells from each coverslip for the cell extension assay.
	<ol>
		<li>Take photomicrographs at 400x magnification of each field of view. Directly count the number of each type of extension for each of the 20 representative cells. 
		NOTE: As such, 20 isolated cells should be examined for each tested condition to ensure reliable results. Filopodia were defined by cellular extensions with a base of 5 microns or less and a single apex. Lobopodia were defined by cellular extensions with a base of 5 to 20 microns of length containing extensions with multiple apices. Lamellipodia were defined by cell extensions that are greater than 20 microns in length and with or without apices. Depending on the tested cell type, the sizes of cell extensions may differ and may require refinement of identifying parameters.</li>
	</ol>
	</li>
	<li>Determine the average number of each cell extension in each cell type (i.e., SW-13, SW-Neo, SW-DDK) across the 6 wells examined.</li>
	<li>Using a one-way analysis of variance (ANOVA) statistical test, compare differences in the average percentage of cell-membrane extensions among the different test cell types.</li>
</ol>
2. Adhesion Assay
<ol>
	<li>Maintain SW-13, SW-Neo, and SW-DKK3 cells in a standard humidified incubator at 37.0 &#176;C and 5% CO2 in growth medium.
	<ol>
		<li>Using a hemocytometer, count and plate 100,000 cells of SW-13, SW-Neo, and SW-DKK3 per well into separate 6-well plates and grow overnight in growth medium.</li>
		<li>Seed 6-well plates for each cell type tested, using one plate for each of the 6 designated time points tested below. 
		NOTE: The time points may vary for different cell types.</li>
	</ol>
	</li>
	<li>After overnight incubation, wash cells with warm PBS once then add 0.5 mL of 1x non-enzymatic cell dissociation solution to each well.
	<ol>
		<li>Swirl the plate to spread the cell dissociation solution. Aspirate the designated plate at defined time points (i.e., 1, 2, 3, 5, 10, and 15 min) and then wash with 1 mL warm PBS solution three times.</li>
		<li>Between washes, gently tap the plates to detach loosely attached cells.</li>
	</ol>
	</li>
	<li>Aspirate any remaining PBS and fix the cells remaining attached to the plate with 1 mL of 3.7% formaldehyde for 10 min.</li>
	<li>Stain cells with 1 mL of 0.05% crystal violet for 30 min then wash excess dye away with 1 mL of deionized water. Titrate wash to promote cell visualization. 
	NOTE: Depending on the level of dye uptake, several additional washes may be needed to promote cell visualization.</li>
	<li>Using a light microscope at 100x magnification, count the remaining attached cells in each well for each plate. 
	NOTE: Use of transparent rulers or marking fine pen guides on the bottom side of the plate will help to avoid repeated counting of the same cells.</li>
	<li>Determine the average number of attached cells per well for each plate.</li>
	<li>Compare the rate of cell detachment between SW-13, SW-Neo, and SW-DDK3 cells over the given time period using a two-way ANOVA statistical test.</li>
</ol>

<ol>
	<li>Libe, R., Fratticci, A., Bertherat, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adrenocortical+cancer:+pathophysiology+and+clinical+management.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+the+Wnt+pathway+in+cancer:+the+emerging+role+of+Dickkopf-3.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collective+cell+migration+in+morphogenesis,+regeneration+and+cancer.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L-type+calcium+channels+regulate+filopodia+stability+and+cancer+cell+invasion+downstream+of+integrin+signalling.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fascin:+Invasive+filopodia+promoting+metastasis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activating+the+nuclear+piston+mechanism+of+3D+migration+in+tumor+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+silencing+of+RASSF1A+deregulates+cytoskeleton+and+promotes+malignant+behavior+of+adrenocortical+carcinoma.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+FOXO1-mediated+dedifferentiation+blocking+role+for+DKK3+in+adrenocortical+carcinogenesis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open+source+software+for+quantification+of+cell+migration,+protrusions,+and+fluorescence+intensities.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+3D+analysis+of+complex+single+border+cell+behaviors+in+coordinated+collective+cell+migration.">Quantitative 3D analysis of complex single border cell behaviors in coordinated collective cell migration.</a> Nat Commun. 8, 14905 (2017).</li></ol>

The authors have nothing to disclose.

Here we describe an in vitro quantitative method to characterize cell extensions with ease, few pit-falls, and reliable reproducibility that can be applied for various test conditions. Moreover, simple, quantifiable adhesion and/or motility assays can be performed simultaneously to correlate potential functional significance of observed alterations in cell membrane extensions.
However, these methods may present some potential limitations. First, the criteria specified here to identify...

Dysregulated WNT signaling plays a critical role in adrenocortical malignancies1. The methods used in this study investigate whether silencing of DKK3, a negative regulator of WNT signaling, represents a dedifferentiation event in the adrenal cortex and promotes tumor formation in the context of cell-extension repertoire changes. DKK3 is a 38 kDa secreted glycoprotein with an N-terminal signal peptide and previous studies have demonstrated that its enforced expression resulted in cell cycle arrest, inhibited aggressive malignant behavior, and reversed epithelial-mesenchymal transition2.
The malignant behavior of adrenocortical carcinoma (ACC) and other cancers is, in part, influenced by the ability of tumor cells to interface with the surrounding surfaces, including the extracellular matrix, which in turn facilitates tumor cell invasion and migration3. The role of specific cell membrane extensions in cancer progression is being increasingly demonstrated in various contexts, primarily via the formation of filopodia. For example, overexpression of L-type calcium channels has been found to induce filopodia formation and promote tumor cell invasion4. Similarly, Fascin, an actin binding protein minimally expressed in normal tissue, is also overexpressed in cancer cells in association with filopodia formation5. Lobopodia formation enables non-malignant fibroblasts to migrate effectively through the extra-cellular matrix, however, it has been shown that fibrosarcoma cells rely on metalloproteinase activity in lieu of lobopodia to facilitate cell migration and invasion6. We have shown that tumor suppressors, including Ras association domain family 1 isoform A (RASSF1A) and DKK3, can function to alter cytoskeletal elements and promote lamellipodia formation and stymie invasive properties7,8.
As such, it is critical to characterize the effects of genes involved in carcinogenesis and their relationship to cell-membrane extension alterations, specifically assessing filopodia, lobopodia, and lamellipodia formation under test conditions. Current state-of-the art techniques include the use of increasingly sophisticated microscopy methods, fluorescent labeling, and/or complex computer algorithms for data acquisition and interpretation. While these methods provide new and powerful analytic tools, their complexity limits their widespread use and adaptability in cell biology experiments. Furthermore, the precise quantification and observation of changes in cell extension morphology is not typically measured9,10. In contrast, we introduce a technique here that accurately quantifies cell extension alterations using standard microscopic techniques and readily adaptable in vitro methods. These methods also quantify each cell extension type simultaneously for each cell analyzed and determine overall changes in the cell membrane extension repertoire. We also show how these changes can relate to cell adhesion properties.
As an experimental example, we will use a previously created cell line of SW-13, designated SW-DDK3, which has been stably transfected with pCMV6-Entry/DKK3 plasmid vectors and constitutively overexpresses DKK3, a differentiating factor in the adrenal gland. Non-transfected SW-13 cells and SW-13 cells stably transfected with empty vector (pCMV6-Entry), designated SW-Neo, will serve as experimental controls.

<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

The cell membrane&#39;s extension repertoire modulates various malignant behaviors of cancer cells, including their adhesive and migratory potentials. The ability to accurately classify and quantify cell extensions and measure the effect on a cell&#39;s adhesive capacity is critical to determining how cell-signaling events impact cancer cell behavior and aggressiveness. Here, we describe the in vitro design and use of a cell extension quantification method in conjunction with an adhesion capacity assay in an established in vitro model for adrenocortical carcinoma (ACC). Specifically, we test the effects of DKK3, a putative tumor suppressor and a pro-differentiation factor, on the membrane extension phenotype of the ACC cell line, SW-13. We propose these assays to provide relatively simple, reliable, and easily interpretable metrics to measures these characteristics under various experimental conditions.

Using the above assays, the effects of DKK3 overexpression on cell extension morphology and cell adhesion properties were tested in the established ACC cell line SW-13, in vitro. Cells overexpressing DKK3 were generated by stably transfecting SW-13 cells with Myc-DDK tagged pCMV6-Entry/DKK3 plasmid vectors and designated as SW-DKK3. Similarly, cells stably transfected with empty vector pCMV6-Entry were created as a transfection and passaging control and defined as SW-Neo. SW-13 c...

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

This study demonstrates the utility and ease of quantitative cell membrane extension measurement and its correlation to adhesive capacity of cells. As a representative example, we show here that Dickkopf-related protein 3 (DKK3) promotes increased lobopodia formation and cell adhesiveness in adrenocortical carcinoma cells in vitro.

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

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Research

JoVE Journal

Cancer Research

8.2K Views.  Yale University School of Medicine. Deze studie toont het nut en het gemak van kwantitatieve celmembraan extensie meting en de correlatie met zelfklevende capaciteit van cellen. Als een representatief voorbeeld, laten we zien hier dat Dickkopf-gerelateerde eiwitten 3 (DKK3) bevordert de vorming van de verhoogde lobopodia en cel hechting in adrenocortical carcinoom cellen in vitro.

Video: 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.

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

Medicine

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.

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. ...

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.

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

Bioengineering

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.

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

Biochemistry

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.

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.

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

Biology

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.

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.

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.

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.

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

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