Name: quantitative analysis of cell edge dynamics during cell spreading
Uploaded: 2021-05-22T15:00:00
Description: Watch this Scientific Journal Video about Quantitative Analysis of Cell Edge Dynamics during Cell Spreading at JoVE.com

This work was supported by the Connaught Fund New Investigator Award to S.P., Canada Foundation for Innovation, NSERC Discovery Grant Program (grants RGPIN-2015-05114 and RGPIN-2020-05881), University of Manchester and University of Toronto Joint Research Fund, and University of Toronto XSeed Program.

1. Cell Seeding
NOTE: The described cell spreading protocol was performed using mouse embryonic fibroblasts (MEFs) expressing PH-Akt-GFP (a fluorescent marker for PIP3/PI(3,4)P2). This cell line was generated by genomically integrating an expression construct for PH-Akt-GFP (Addgene #21218) by CRISPR-mediated gene editing. However, other fluorescent markers that are expressed transiently or integrated in the genome can also be used in this assay. For optimal image segmentat...

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The described cell spreading assay allows for the continuous tracking of morphological changes (e.g., cell size and shape) and cell edge movements (i.e., protrusion speed and retraction frequency), which are features missing in most cell spreading protocols19,24. While commonly used end-point cell spreading assays allow for the determination of cell spreading speed, these assays fail to resolve the temporal dynamics of cell edge movements. The l...

Lamellipodial protrusions are prominent cytoskeletal structures formed at the front of a migrating cell. In lamellipodia, polymerization of actin with the aid of the Arp2/3 complex and formins&#160;creates a fast-growing branched actin meshwork that pushes against the plasma membrane1,2. The pushing force generated by the actin meshwork physically propels the cell forward1,3,4,5. Depletion of the Arp2/3 complex or disruption of signaling pathways essential for lamellipodial protrusions often impair cell migration6, 7. Although migration of lamellipodia-deficient cells has also been reported8,9, the importance of lamellipodia in cell migration is evident as depletion of this protrusive structure perturbs the cell&#39;s ability to move through&#160;complex biological microenvironments6,10.
A major hindrance to understanding the regulation of lamellipodia in migrating cells is the natural variability in lamellipodial protrusion kinetics, size, and shape11,12,13,14. Furthermore, recent studies have demonstrated that lamellipodia exhibit complex protrusive behaviors, including fluctuating, periodic, and accelerating protrusions14,15. Compared to the highly variable lamellipodia of migrating cells6,16, lamellipodia formed during cell spreading are more uniform12. Since the protrusive activity of spreading and migrating cells is driven by identical macromolecular assemblies, which include a branched actin network, contractile actomyosin bundles, and integrin-based cell-matrix adhesions17,18, spreading cells have been widely used as a model for investigating the regulation of lamellipodia dynamics.
Cell spreading is a dynamic mechanochemical process where a cell in suspension first adheres to a substrate through integrin-based adhesions17,19,20 and then spreads by extending actin-based protrusions21,22,23. During the spreading phase, lamellipodia emanating from the cell body protrude isotropically and persistently with little to no retraction or stalling12. The most commonly used cell spreading protocols are endpoints assays, where spreading cells are fixed at various times after plating19,24. These assays, although quick and simple, are limited in their diagnostic power to detect changes in the dynamic features of lamellipodia. To determine the molecular mechanisms that control lamellipodia dynamics, the Sheetz group pioneered the use of quantitative analysis of live spreading cells and uncovered many fundamental properties of cell edge protrusions11,12,22. These studies have demonstrated that the live-cell spreading assay is a robust and powerful technique in the toolbox of a cell biology laboratory. Despite that, a detailed protocol and open-source computational tool for a live-cell spreading assay are currently unavailable for the cell biology community. To this end, our protocol outlines the procedures of imaging live spreading cells and provides an automated image analysis tool. To validate this method, we used Arp2/3 inhibition as an experimental treatment and showed that inhibiting the function of the Arp2/3 complex did not arrest cell spreading but caused a significant reduction in cell protrusion speed, as well as the stability of cell edge protrusions, giving rise to jagged cell edges. These data demonstrate that the combination of live-cell imaging and automated image analysis is a useful tool for analyzing cell edge dynamics and identifying molecular components that regulate lamellipodia.

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			<td>0.05% Trypsin (0.05%), 0.53 mM EDTA</td>
			<td>Wisent Bioproducts</td>
			<td>325-042-CL</td>
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			<td>10.0 cm Petri Dish, Polystyrene, TC Treated, Vented</td>
			<td>Starstedt</td>
			<td>83.3902</td>
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			<td>15 mL High Clarity PP Centrifuge Tube, Conical Bottom, with Dome Seal Screw Cap, Sterile</td>
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			<td>352097</td>
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			<td>1-Well Chamlide CMS for 22 mm x 22 mm Coverslip</td>
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			<td>CM-S22-1</td>
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			<td>35 mm TC-treated Easy-Grip Style Cell Culture Dish</td>
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			<td>DMEM/F-12, HEPES, No Phenol Red</td>
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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.

The above protocol describes the experimental procedures for the live-cell imaging of spreading cells and a computational tool for the quantitative analysis of cell spreading dynamics. The computational tool can be used in a low- or high-throughput format to identify the molecular players regulating the actin polymerization machinery at the cell leading edge.
The schematic representation of the experimental procedures is depicted in Figure 1. The cell spreading as...

Watch this Scientific Journal Video about Quantitative Analysis of Cell Edge Dynamics during Cell Spreading at JoVE.com

Quantitative Analysis of Cell Edge Dynamics during Cell Spreading

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 ...

The protocol described in this video will define the experimental procedures required for the life-size imaging of spreading cells and the use of a computational tool that quantifies cells spreading dynamics in an unbiased and automated fashion. The cell spreading asset allows for the continuous tracking of cell edge movement and morphological changes during cell spreading, which is a feature that is missing in most existing cell spreading assays. As well, this protocol implements the use of automated computer processing and analysis, providing information on cell circularity, area and protrusion retraction cycles of spreading cells.Such automated processing reduces bias in data analysis and provides a robust method of analyzing cell spreading dynamics for a large number of cells. When combined with drug treatments or gene science and techniques, this protocol is amenable to large-scale speeds of molecular players regulating cell protrusions. For the given protocol, The cells used are mouse embryonic fibroblasts that genetically encode PH-Akt-GFP, which allows for the fluorescent tagging of the plasma membrane.Prior to the beginning of the cell spreading assay, culture a dish of cells to 90%confluency. Once the cells have attained the proper confluency, flame a 22 by 22 millimeter cover slip and place it into a 35 millimeter cell culture dish. Coat the cover slip with fibronectin that has been diluted in PBS to a concentration of 2.5 micrograms per milliliter.Place the dish with the cover slip into a 37 degree incubator for one hour. After one hour aspirate the fibronectin making sure not to touch the cover slip with the pipette tip. Wash the dish with PBS by gently pipetting around the cover slip two to three times.For cell seeding, begin by aspirating the cell culture media from the dish of cells. Afterwards, wash the dish with warm PBS. Add 650 microliters of trypsin EDTA to the dish and tilt the dish to evenly distribute the enzyme.Place the dish with the trypsin into the incubator for one minute. After incubation you will first need to add 10 milliliters of cell culture media into a centrifuge tube. Next, quickly add another 10 milliliters of media into the dish in order to quench the trypsin.To dilute the cells that will be seeded onto the cover slip, pipette one milliliter of the dish's contents into the centrifuge tube. From the tube, pipette approximately 500 to 1, 000 microliters of diluted cells into the dish with the cover slip. Ensure that the cover slip is at a 10%confluency or 50, 000 cells per milliliter and adjust the volume of diluted cells as needed.The purpose of these cells is to establish a plane of focus during image acquisition. With the remaining cells in the dish, passage one fifth of the cells into one small dish per treatment. These will be the cells that will be analyzed for spreading dynamics.Place the passage dishes and the cover slip dish into an incubator for eight to 24 hours. To test the importance of Arp2/3 for cell spreading, first add five milliliters of cell culture media into a control and a treatment centrifuge tube. Next add 20 milliliters of dmem that is lacking phenol red into each of two larger centrifuge tubes.Pipette either the drug CK-666 which is an inhibitor of the Arp2/3 complex or the controlled treatment such as DMSO into the small and large tubes. To begin pharmacological treatment of the cells, remove the passage dishes that were incubating overnight. Aspirate the cell culture media from all of the dishes and wash the dishes with warm PBS.Take the small tubes containing either CK-666 or control supplemented media and add the contents of the relevant tube into each of the passage dishes. Label each of the dishes with the correct drug treatment and then place the dishes back into the incubator for an additional hour After one hour incubation, aspirate the drug supplemented media from each of the dishes. Next, add warm PBS to all of the dishes in order to thoroughly remove all of the remaining phenol red media.Add 230 microliters of trypsin EDTA and place the dishes into an incubator for one minute. Remove the trypsinized dishes from the incubator and proceed to add five milliliters of the drug supplemented dmem that is lacking phenol red into a tube designated as tube B.Then add another five milliliters of the same media into the relevant dish to quench the trypsin. Pipette the media up and down several times in order to detach all of the cells from the dish.Transfer all of the contents of the dish into another tube that has already been designated as tube A.In order to prepare a cell dilution that is appropriate for imaging, transfer one milliliter of cells from tube A into tube B.Repeat all of the dilution steps for each treatment. Place all of the tubes into the incubator for 45 minutes to allow cells to recover from trypsinization. Ensure that all parts of a cell magnetic chamber that can accommodate a 22 by 22 millimeter cover slip have been thoroughly cleaned before use.Remove the dish with the cover slip from the incubator. Aspirate the cell culture media and wash the cover slip with warm PBS. Remove the cover slip using a pair of forceps and gently lay the cover slip onto the bottom plate of the magnetic chamber.Next pick up the silicone gasket and place it on top of the cover slip. Attach the main body of the magnetic chamber onto the bottom plate. Add one milliliter of the dmem lacking phenol red corresponding to the relevant treatment to the magnetic chamber.Take a lint-free tissue and carefully dab the enclosure between the main body and the bottom plate in order to check for any leaks. To complete the magnetic chamber, lower the transparent cover onto the main body. Lastly wipe the bottom of the cover slip with a tissue sprayed with water and another sprayed with 70%ethanol.Preheat the stage top incubator of a confocal microscope to 37 degrees. Place the magnetic chamber on top of the objective. Prior to acquiring spreading dynamics, set focus to the already polarized cells in the green fluorescent channel.This ensures that the spreading cells will be in focus once they attach to the cover slip. Remove the transparent cover of the magnetic chamber and pipette 500 microliters from tube B that was removed from the incubator. Place the transparent cover back on top.To identify cells ideal for cell spreading analysis search for green halos which represent cells that have yet to attach to the cover slip or are in the earliest stages of attachment. Acquire images and save the files. After having completed the image acquisition of cell spreading, begin by running a compatible Python IDE such as Spyder.For computational analysis of cell spreading dynamics, locate and open the cell spreading GUI file provided in the relevant script packages. Press the run button which will open the analysis GUI panel in the background. The GUI houses all of the settings needed for analysis.To analyze cell area and circularity, input the acquisition file and the necessary settings in the cell spread area tab. Adjustments will have to be made depending on cell type and image acquisition parameters. After pressing run, the script will create a cell circularity and area versus time plot for all spreading cells.For kymograph data, press the kymograph generator and analysis tab. This analysis requires input files to be cropped single cell image stacks. After inserting all of the settings and pressing run, the script will create multiple kymographs for the given cell.On the left are representative cells from the DMSO and CK-666 treatments, automatically segmented using the provided script. As opposed to the isotropic and highly circular morphology demonstrated by control cells, Arp2/3 inhibited cells exhibit decreased circularity. Additionally, the automatically generated kymographs provide temporal resolution that is critical to understanding the dynamics of protrusions.Control cells protrude persistently with little to no retractions. In contrast, Arp2/3 inhibition disrupts the stability of protrusions as indicated by the increase in retraction events throughout spreading. This videos should provide you with the understanding of how to properly seed cells, perform pharmacological treatments and prepare imaging and computational tools necessary for the quantification of cell spreading dynamics.This protocol can be further combined with cytoskeleton fluorescent imaging and migration assays to identify the molecular players governing cell protrusions. Thank you for watching and good luck with your experiments.

quantitative-analysis-of-cell-edge-dynamics-during-cell-spreading

Research

JoVE Journal

Biology

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique advantage of FSM is its ability to capture the movement and turnover kinetics (assembly/disassembly) of the F-actin network within living cells. This technique is particularly useful in deriving quantitative measurements of F-actin dynamics when paired with computer vision software (qFSM).  We describe  the selection, microinjection and visualization of fluorescent actin probes in living cells. Importantly, similar procedures are applicable to visualizing other macomolecular assemblies. FSM has been demonstrated for microtubules, intermediate filaments, and adhesion complexes.  

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Determining Cell Number During Cell Culture using the Scepter Cell Counter

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and accuracy. Cell counts are important for monitoring cell health and proliferation rate, assessing immortalization or transformation, seeding cells for subsequent experiments, transfection or infection, and preparing for cell-based assays. It is important that cell counts be accurate, consistent, and fast, particularly for quantitative measurements of cellular responses.
Despite this need for speed and accuracy in cell counting, 71% of 400 researchers surveyed1 who count cells using a hemocytometer. While hemocytometry is inexpensive, it is laborious and subject to user bias and misuse, which results in inaccurate counts. Hemocytometers are made of special optical glass on which cell suspensions are loaded in specified volumes and counted under a microscope. Sources of errors in hemocytometry include: uneven cell distribution in the sample, too many or too few cells in the sample, subjective decisions as to whether a given cell falls within the defined counting area, contamination of the hemocytometer, user-to-user variation, and variation of hemocytometer filling rate2.
To alleviate the tedium associated with manual counting, 29% of researchers count cells using automated cell counting devices; these include vision-based counters, systems that detect cells using the Coulter principle, or flow cytometry1. For most researchers, the main barrier to using an automated system is the price associated with these large benchtop instruments1.
The Scepter cell counter is an automated handheld device that offers the automation and accuracy of Coulter counting at a relatively low cost. The system employs the Coulter principle of impedance-based particle detection3 in a miniaturized format using a combination of analog and digital hardware for sensing, signal processing, data storage, and graphical display. The disposable tip is engineered with a microfabricated, cell- sensing zone that enables discrimination by cell size and cell volume at sub-micron and sub-picoliter resolution. Enhanced with precision liquid-handling channels and electronics, the Scepter cell counter reports cell population statistics graphically displayed as a histogram.

The Scepter Cell Counter is a handheld automated device that can be used to count cells, monitor cell diameter and volume, and be used to check the health and quality of cellular populations from one culture to the next.

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

A Quantitative Fitness Analysis Workflow

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. QFA can be applied to focused observations of single cultures but is most useful for genome-wide genetic interaction or drug screens investigating up to thousands of independent cultures. The central experimental method is the inoculation of independent, dilute liquid microbial cultures onto solid agar plates which are incubated and regularly photographed. Photographs from each time-point are analyzed, producing quantitative cell density estimates, which are used to construct growth curves, allowing quantitative fitness measures to be derived. Culture fitnesses can be compared to quantify and rank genetic interaction strengths or drug sensitivities. The effect on culture fitness of any treatments added into substrate agar (e.g. small molecules, antibiotics or nutrients) or applied to plates externally (e.g. UV irradiation, temperature) can be quantified by QFA.
The QFA workflow produces growth rate estimates analogous to those obtained by spectrophotometric measurement of parallel liquid cultures in 96-well or 200-well plate readers. Importantly, QFA has significantly higher throughput compared with such methods. QFA cultures grow on a solid agar surface and are therefore well aerated during growth without the need for stirring or shaking.
QFA throughput is not as high as that of some Synthetic Genetic Array (SGA) screening methods5,6. However, since QFA cultures are heavily diluted before being inoculated onto agar, QFA can capture more complete growth curves, including exponential and saturation phases3. For example, growth curve observations allow culture doubling times to be estimated directly with high precision, as discussed previously1.
Here we present a specific QFA protocol applied to thousands of S. cerevisiae cultures which are automatically handled by robots during inoculation, incubation and imaging. Any of these automated steps can be replaced by an equivalent, manual procedure, with an associated reduction in throughput, and we also present a lower throughput manual protocol. The same QFA software tools can be applied to images captured in either workflow.
We have extensive experience applying QFA to cultures of the budding yeast S. cerevisiae but we expect that QFA will prove equally useful for examining cultures of the fission yeast S. pombe and bacterial cultures.

Quantitative Fitness Analysis (QFA) is a complementary series of experimental and computational methods for estimating microbial culture fitnesses. QFA estimates the effect of genetic mutations, drugs or other applied treatments on microbe growth. Experiments scaling from focussed analysis of single cultures to thousands of parallel cultures can be designed.

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. ...

Surface Spreading and Immunostaining of Yeast Chromosomes

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution of chromatin-bound proteins. Surface spreading of yeast nuclei results in expansion of chromatin without loss of bound proteins. A method for surface spreading balances fixation of DNA bound proteins with detergent treatment. The method demonstrated is slightly modified from that described by Josef Loidl and Franz Klein1,2. The method has been used to characterize the localization of many chromatin-bound proteins at various stages of the mitotic cell cycle, but is especially useful for the study of meiotic chromosome structures such as meiotic recombinosomes and the synaptonemal complex. We also describe a modification that does not require use of Lipsol, a proprietary detergent, which was called for in the original procedure, but no longer commercially available. An immunostaining protocol that is compatible with the chromosome spreading method is also described.

A method for surface-spreading chromosomes from budding yeast is presented. This method is derived from a method previously described by Loidl and Klein. In addition, we demonstrate a procedure for immunostaining of spread chromosomes.

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution ...

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells. Cells expressing fluorescently tagged tubulin are adhered to assembled slide chambers, allowing for stable time-lapse image acquisition. A detailed guide for high-speed, four-dimensional image acquisition is also provided, as well as a protocol for quantifying the properties of dynamic microtubules in confocal image stacks. This method, combined with conventional yeast genetics, provides an approach that is uniquely suited for quantitatively assessing the effects of microtubule regulators or mutations that alter the activity of tubulin subunits.

Budding yeast is an advantageous model for studying microtubule dynamics in vivo due to its powerful genetics and the simplicity of its microtubule cytoskeleton. The following protocol describes how to transform and culture yeast cells, acquire confocal microscopy images, and quantitatively analyze microtubule dynamics in living yeast cells.

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells ...

Live Cell Fluorescence Microscopy to Observe Essential Processes During Microbial Cell Growth

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of proteins which are essential for bacterial survival. A series of target-specific dyes can be used as probes to better understand these processes. Staining with lipophilic dyes enables the observation of membrane structure, visualization of lipid microdomains, and detection of membrane blebs. Use of fluorescent-d-amino acids (FDAAs) to probe the sites of peptidoglycan biosynthesis can indicate potential defects in cell wall biogenesis or cell growth patterning. Finally, nucleic acid stains can indicate possible defects in DNA replication or chromosome segregation. Cyanine DNA stains label living cells and are suitable for time-lapse microscopy enabling real-time observations of nucleoid morphology during cell growth. Protocols for cell labeling can be applied to protein depletion mutants to identify defects in membrane structure, cell wall biogenesis, or chromosome segregation. Furthermore, time-lapse microscopy can be used to monitor morphological changes as an essential protein is removed and can provide additional insights into protein function. For example, the depletion of essential cell division proteins results in filamentation or branching, whereas the depletion of cell growth proteins may cause cells to become shorter or rounder. Here, protocols for cell growth, target-specific labeling, and time-lapse microscopy are provided for the bacterial plant pathogen Agrobacterium tumefaciens. Together, target-specific dyes and time-lapse microscopy enable characterization of essential processes in A. tumefaciens. Finally, the protocols provided can be readily modified to probe essential processes in other bacteria.

Understanding the function of essential processes in bacteria is challenging. Fluorescence microscopy with target-specific dyes can provide key insights into microbial cell growth and cell cycle progression. Here, Agrobacterium tumefaciens is used as a model bacterium to highlight methods for live cell imaging for characterization of essential processes.

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of ...

Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator and that it is possible to monitor and quantify single cells along several cell cycles. We describe the full protocol used to monitor and quantify a HeLa cell culture for 2.7 days. First, cell culture acquisition is performed with a lens-free video microscope, and then the data is analyzed following a four-step process: multi-wavelength holographic reconstruction, cell-tracking, cell segmentation and cell division detection algorithms. As a result, we show that it is possible to gather a dataset featuring more than 10,000 cell cycle tracks and more than 2 x 106 cell morphological measurements.

Lens-free video microscopy enables us to monitor cell cultures directly inside the incubator. Here we describe the full protocol used to acquire and analyze a 2.7 day long acquisition of cultured HeLa cells, leading to a dataset of 2.2 x 106 measurements of individual cell morphology and 10584 cell cycle tracks.

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator ...

Micromanipulation Techniques Allowing Analysis of Morphogenetic Dynamics and Turnover of Cytoskeletal Regulators

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how fluorescent recovery after photobleaching (FRAP) and photoactivation techniques can be used to study the spatiotemporal dynamics of proteins in subcellular locations. We also show how these techniques enable straightforward determination of various parameters linked to actin cytoskeletal regulation and cell motility. Moreover, the microinjection of cells is additionally described as an alternative treatment (potentially preceding or complementing the aforementioned photomanipulation techniques) to trigger instantaneous effects of translocated proteins on cell morphology and function. Micromanipulation such as protein injection or local application of plasma membrane-permeable drugs or cytoskeletal inhibitors can serve as powerful tool to record immediate consequences of a given treatment on cell behavior at the single cell and subcellular level. This is exemplified here by immediate induction of lamellipodial cell edge protrusion by the injection of recombinant Rac1 protein, as established a quarter-century ago. In addition, we provide a protocol for determining the turnover of enhanced green fluorescent protein (EGFP)-VASP, an actin filament polymerase prominently accumulating at lamellipodial tips of B16-F1 cells, employing FRAP and including associated data analysis and curve fitting. We also present guidelines for estimating the rates of lamellipodial actin network polymerization, as exemplified by cells expressing EGFP-tagged &#946;-actin. Finally, instructions are given for how to investigate the rates of actin monomer mobility within the cell cytoplasm, followed by actin incorporation at sites of rapid filament assembly, such as the tips of protruding lamellipodia, using photoactivation approaches. None of these protocols is&#160;restricted to components or regulators of the actin cytoskeleton, but can easily be extended to explore in analogous fashion the spatiotemporal dynamics and function of proteins in various different subcellular structures or functional contexts.

We describe how micro- and photomanipulation techniques such as FRAP and photoactivation enable the determination of motility parameters and the spatiotemporal dynamics of proteins within migrating cells. Experimental readouts include subcellular dynamics and turnover of motility regulators or of the underlying actin cytoskeleton.