Name: combining mitotic cell synchronization and high resolution confocal microscopy to study the role of multifunctional cell cycle proteins during mitosis
Uploaded: 2017-12-05T14:00:00
Description: Watch this Scientific Journal Video about Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis at JoVE

We are grateful to Dr. Kozo Tanaka of Tohoku University, Japan for sharing HeLa cells stably expressing mCherry-Histone H2B and GFP-&#945;-tubulin. This work was supported by an NCI grant to DV (R00CA178188) and by start-up funds from Northwestern University.

1. Double Thymidine Block and Release: Reagent Preparations
<ol>
	<li>Make 500 mL Dulbecco&#39;s modified Eagle medium (DMEM) medium supplemented with 10% FBS, penicillin, and streptomycin.</li>
	<li>Make 100 mM stock of thymidine in sterile water and store in aliquots at -80 &#176;C.</li>
</ol>
2. Protocol for Fixed Cell Imaging of Mitotic Progression (Figure 1A)
<ol>
	<li>On day 1, seed ~2 x 105 HeLa cells into the wells of a 6-well plate with a cover...

<ol>
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Pharmacol. 72 (11), 1396-1404 (2006).</li><li>Amon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synchronization+procedures.">Synchronization procedures.</a> Methods Enzymol. 351, 457-467 (2002).</li><li>Cooper, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rethinking+synchronization+of+mammalian+cells+for+cell+cycle+analysis.">Rethinking synchronization of mammalian cells for cell cycle analysis.</a> Cell Mol. Life Sci. 60 (6), 1099-1106 (2003).</li><li>Whitfield, M. L., Sherlock, G., Saldanha, A. J., Murray, J. I., Ball, C. A., Alexander, K. E., Matese, J. C., Perou, C. M., Hurt, M. M., Brown, P. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+evaluation+of+the+double+thymidine+block+for+synchronizing+mammalian+cells+at+the+G1-S+border.">An evaluation of the double thymidine block for synchronizing mammalian cells at the G1-S border.</a> Exp. Cell Res. 68 (1), 163-168 (1971).</li><li>Martin-Lluesma, S., Stucke, V. M., Nigg, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Hec1+in+spindle+checkpoint+signaling+and+kinetochore+recruitment+of+Mad1/Mad2.">Role of Hec1 in spindle checkpoint signaling and kinetochore recruitment of Mad1/Mad2.</a> Science. 297, 2267-2270 (2002).</li><li>Brouwers, N., Mallol Martinez, N., Vernos, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Kif15+and+its+novel+mitotic+partner+KBP+in+K-fiber+dynamics+and+chromosome+alignment.">Role of Kif15 and its novel mitotic partner KBP in K-fiber dynamics and chromosome alignment.</a> PLoS One. 12 (4), e0174819 (2017).</li><li>Dou, Z., 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=Dynamic+localization+of+Mps1+kinase+to+kinetochores+is+essential+for+accurate+spindle+microtubule+attachment.">Dynamic localization of Mps1 kinase to kinetochores is essential for accurate spindle microtubule attachment.</a> Proc Natl Acad Sci USA. 112 (33), E4546-E4555 (2015).</li><li>Chan, Y. 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The most critical advantage of double thymidine synchronization is that it provides an increased sample size of mitotic cells in a short time window with many of these cells entering mitosis in unison, thus also enabling analyses of chromosome alignment, bipolar spindle formation, and chromosome segregation with much higher efficiency.
Many regulatory protein complexes and signaling pathways are devoted to ensuring normal progression through mitosis and deregulation of this process may led to ...

In the cell cycle, cells undergo a series of highly regulated and temporally controlled events for the accurate duplication of their genome and proliferation. In mammals, the cell cycle consists of interphase and M-phase. In interphase, which consists of three stages- G1, S, and G2, the cell duplicates its genome and undergoes growth that is necessary for normal cell cycle progression1,2. In the M-phase, which consists of mitosis (prophase, prometaphase, metaphase, anaphase, and telophase) and cytokinesis, a parental cell produces two genetically identical daughter cells. In mitosis, sister chromatids of duplicated genome are condensed (prophase) and are captured at their kinetochores by microtubules of the assembled mitotic spindle (prometaphase), that drives their alignment at the metaphase plate (metaphase) followed by their equal segregation when sister chromatids are split toward and transported to opposite spindle poles (anaphase). The two daughter cells are physically separated by the activity of an actin-based contractile ring (telophase and cytokinesis). The kinetochore is a specialized proteinaceous structure which assembles at the centromeric region of chromatids and serve as attachment sites for spindle microtubules. Its main function is to drive chromosome capture, alignment, and aid in correcting improper spindle microtubule attachment, while mediating the spindle assembly checkpoint to maintain the fidelity of chromosome segregation3,4.
The technique of cell synchronization serves as an ideal tool for understanding the molecular and structural events involved in cell cycle progression. This approach has been used to enrich cell populations at specific phases for various types of analyses, including profiling of gene expression, analyses of cellular biochemical processes, and detection of subcellular localization of proteins. Synchronized mammalian cells can be used not only for the study of individual gene products, but also for approaches involving analysis of whole genomes including microarray analysis of gene expression5, miRNA expression patterns6, translational regulation7, and proteomic analysis of protein modifications8. Synchronization can also be used to study the effects of gene expression or protein knock-down or knock-out, or of chemicals on cell cycle progression.
Cells can be synchronized at the different stages of the cell cycle. Both physical and chemical methods are widely used for cell synchronization. The most important criteria for cell synchronization are that synchronization should be noncytotoxic and reversible. Because of the potential adverse cellular consequences of synchronizing cells by pharmacological agents, chemical-dependent methods can be advantageous for studying key cell cycle events. For example, hydroxyurea, amphidicolin, mimosine, and lovastatin, can be used for cell synchronization at G1/S phase but, because of their effect on the biochemical pathways they inhibit, they activate cell cycle checkpoint mechanisms and kill an important fraction of the cells9,10. On the other hand, feedback inhibition of DNA replication by adding thymidine to the growth media, known as &#34;thymidine block&#34;, can arrest the cell cycle at certain points11,12,13. Cells can also be synchronized at G2/M phase by treating with nocodazole and RO-33069,14.Nocodazole, which prevents microtubule assembly, has a relatively high cytotoxicity. Moreover, nocodazole-arrested cells can return to interphase precociously by mitotic slippage. Double thymidine block arrest cells at G1/S phase and after release from the block, cells are found to proceed synchronously through G2 and into mitosis. The normal progression of the cell cycle for cells released from thymidine block can be observed under high resolution confocal microscopy by either cell fixation or live imaging. The effect of perturbation of mitotic proteins can be studied specifically when cells enter and proceed through mitosis after release from double thymidine block. Cdt1, a multifunctional protein, is involved in DNA replication origin licensing in the G1 phase and is also required for kinetochore microtubule attachments during mitosis15. To study the function of Cdt1 during mitosis, one needs to adopt a method that avoids the effect of its depletion on replication licensing during G1 phase, while at the same time effecting its depletion specifically during the G2/M phase only. Here, we present detailed protocols based on the double thymidine block to study the mitotic role of proteins performing multiple functions during different stages of the cell cycle by both fixed and live-cell imaging.

<table>
	<tbody>
		<tr>
			<td>DMEM (1X)</td>
			<td>Life Technologies</td>
			<td>11965-092</td>
			<td>Store at 4 degree C</td>
		</tr>
		<tr>
			<td>DPBS (1X)</td>
			<td>Life Technologies</td>
			<td>14190-144</td>
			<td>Store at 4 degree C</td>
		</tr>
		<tr>
			<td>Leibovitz&#8217;s (1X) L-15 medium</td>
			<td>Life Technologies</td>
			<td>21083-027</td>
			<td>Store at 4 degree C</td>
		</tr>
		<tr>
			<td>Serum reduced medium (Opti-MEM)</td>
			<td>Life Technologies</td>
			<td>319-85-070</td>
			<td>Store at 4 degree C</td>
		</tr>
		<tr>
			<td>Penicillin and streptomycin</td>
			<td>Life Technologies</td>
			<td>15070-063 (Pen Strep)</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr>
			<td>Dharmafect2</td>
			<td>GE Dharmacon</td>
			<td>T-2002-02</td>
			<td>Store at 4 degree C</td>
		</tr>
		<tr>
			<td>Thymidine</td>
			<td>MP Biomedicals LLC</td>
			<td>103056</td>
			<td>Dissolved in sterile distiled water</td>
		</tr>
		<tr>
			<td>Cdt1 siRNA</td>
			<td>Life Technologies</td>
			<td></td>
			<td>Ref 10</td>
		</tr>
		<tr>
			<td>Hec1 siRNA</td>
			<td>Life Technologies</td>
			<td></td>
			<td>Ref 13</td>
		</tr>
		<tr>
			<td>HeLa cells expressing GFP-H2B</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HeLa cells expressing GFP-&#945;-tubulin and mCherry H2B</td>
			<td>Generous gift from Dr. Kozo Tanaka of Tohoku university, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde solution</td>
			<td>Sigma-Aldrich Corporation</td>
			<td>F8775</td>
			<td>Toxic, needs caution</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich Corporation</td>
			<td>D9542</td>
			<td>Toxic, needs caution</td>
		</tr>
		<tr>
			<td>Mouse anti-&#945;-tubulin</td>
			<td>Santa Cruz Biotechnology</td>
			<td>Sc32293</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr>
			<td>Rabbit ant-Zwint1</td>
			<td>Bethyl</td>
			<td>A300-781A</td>
			<td>1:400 dilution</td>
		</tr>
		<tr>
			<td>Mouse anti-phospho-&#947;H2AX (Ser139)</td>
			<td>Upstate Biotechnology</td>
			<td>05-626, clone JBW301</td>
			<td>1:300 dilution</td>
		</tr>
		<tr>
			<td>Alexa 488</td>
			<td>Jackson ImmunoResearch</td>
			<td></td>
			<td>1:250 dilution</td>
		</tr>
		<tr>
			<td>Rodamine Red-X</td>
			<td>Jackson ImmunoResearch</td>
			<td></td>
			<td>1:250 dilution</td>
		</tr>
		<tr>
			<td>BioLite 6 well multidish</td>
			<td>Thermo Fisher Scientific</td>
			<td>130184</td>
			<td></td>
		</tr>
		<tr>
			<td>35MM Glass bottom dish</td>
			<td>MatTek Corporation</td>
			<td>P35GCOL-1.5-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Eclipse TiE inverted microscope</td>
			<td>Nikon Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spinning disc for confocal</td>
			<td>Yokagawa</td>
			<td>CSU-X1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra 888 EM-CCD Camera</td>
			<td>Andor</td>
			<td>iXon Ultra EMCCD</td>
			<td></td>
		</tr>
		<tr>
			<td>4 wave length laser</td>
			<td>Agilent Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Incubation System for Microscopes</td>
			<td>Tokai Hit</td>
			<td>TIZB</td>
			<td></td>
		</tr>
		<tr>
			<td>NIS-elements software</td>
			<td>Nikon Instruments</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

combining mitotic cell synchronization and high resolution confocal microscopy to study the role of multifunctional cell cycle proteins during mitosis

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The synchronization of cell populations at specific stages of the cell cycle has been found to be very useful in such experimental endeavors. Synchronization of cells by treatment with chemicals that are relatively less toxic can be advantageous over the use of pharmacological inhibitory drugs for the study of consequent cell cycle events and to obtain specific enrichment of selected mitotic stages. Here, we describe the protocol for synchronizing human cells at different stages of the cell cycle, including both in S phase and M phase with a double thymidine block and release procedure for studying the functionality of mitotic proteins in chromosome alignment and segregation. This protocol has been extremely useful for studying the mitotic roles of multifunctional proteins which possess established interphase functions. In our case, the mitotic role of Cdt1, a protein critical for replication origin licensing in G1 phase, can be studied effectively only when G2/M-specific Cdt1 can be depleted. We describe the detailed protocol for depletion of G2/M-specific Cdt1 using double thymidine synchronization. We also explain the protocol of cell fixation, and live cell imaging using high resolution confocal microscopy after thymidine release. The method is also useful for analyzing the function of mitotic proteins under both physiological and perturbed conditions such as for Hec1, a component of the Ndc80 complex, as it enables one to obtain large sample sizes of mitotic cells for fixed and live cell analysis as we show here.&#160;&#160;

Study of mitotic progression and microtubule stability in cells fixed after release from double thymidine block 
Cdt1 is involved in licensing of DNA replication origins in the G1 phase. It is degraded during S phase but re-accumulates in G2/M phase. To study its role in mitosis, the endogenous Cdt1 needs to be depleted specifically at the G/M phase using the most suitable cell synchronization technique, the double thymidine block15. Cells wer...

Watch this Scientific Journal Video about Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis at JoVE

Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis

We present a protocol for double thymidine synchronization of HeLa cells followed by analysis using high resolution confocal microscopy. This method is key to obtaining large number of cells that proceed synchronously from S phase to mitosis, enabling studies on mitotic roles of multifunctional proteins which also possess interphase functions.

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The ...

The overall goal of this procedure is to use double thymidine synchronization and high-resolution confocal microscopy to study the mitotic roles of multifunctional proteins that may possess critical interphase functions. This method can help answer key questions in the cell biology field, about how to delineate the normal functions of proteins involved in cell cycle and mitosis. The main advantages of this technique are that, the cells can maintain their normal physiological behavior and that this method is also very easy to perform.Demonstrating this procedure will be Dr.Mohammed Amin, a post-doc from my laboratory, who is an expert in the use of this method. For fixed cell imaging of mitotic cell progression, begin by seeding approximately two times 10 to the fifth HeLa cells into each well of a six-well plate, containing a 70%ethanol and UV sterilized cover slip and two milliliters of DMEM medium. After 24 hours in a cell culture incubator, block the cells with two milliliters of freshly diluted thymidine and fresh medium per well, and return the plate to the incubator for another 18 hours.The next day, wash the cells two times with two milliliters of PBS, and one time with fresh 37 degree Celsius medium. Return the cells to the incubator for nine hours to release the cells from the block, followed by the addition of another two milliliters of thymidine-supplemented medium per well. After the second blocking incubation, wash the cells two times with two milliliters of PBS, and one time with two milliliters of fresh 37 degree Celsius medium, and add 100 nanomolar of freshly prepared siRNA to the appropriate wells.After nine to 10 hours, aspirate the supernatant and fix the cells onto the cover slips with four percent paraformaldehyde for 20 minutes at room temperature. After washing with PBS, permeabilize the fixed cells with 0.5%detergent for 10 minutes at room temperature, followed by two five-minute washes in PBS. Block the cells with one percent BSA in PBS for one hour at room temperature.Then label the cells with 50 microliters of the primary antibodies of interest for one hour at 37 degrees Celsius. At the end of the incubation, wash the cells three times in PBS, and label them with 50 microliters of the appropriate secondary antibodies of interest. After one hour at room temperature, wash the cells two times in PBS for five minutes each wash, and label the cells with DAPI for five minutes at room temperature.Follow the DAPI staining with two five-minute washes in PBS and use the appropriate mounting medium to mount the cover slips on individual clear microscope slides. Then, using 60 or 100 X 1.4 numerical aperture plan apochromatic DIC oil immersion objectives mounted on an inverted high resolution confocal microscope equipped with an appropriate camera, acquire images of the immunostained proteins in Z stacks of 0.2 micrometer thickness. For live cell imaging of mitotic cell progression, seed approximately 0.5 to one times 10 to the fifth HeLa cells stably expressing mCherry H2B and GFP alpha-tubulin into 35-milliliter glass-bottom dishes containing 1.5 milliliters of DMEM medium.Grow the cells in a cell culture incubator for 24 hours. Then, thymidine block the cells two times as just demonstrated, this time washing the cells two times with two milliliters of PBS and one time with one milliliter of pre-warmed DMEM medium at the end of each thymidine block and treating the cells with siRNA after the first block during the first thymidine washout. Grow the cells in fresh pre-warmed Leibovitz's medium, supplemented with 10%FBS and 20 millimolar HEPES for eight hours to release the cells from the second block.Then place the first plate in the temperature-controlled chamber of a high-resolution confocal microscope, and use the 60 X objective and bright field imaging to bring the cells into focus. When the cells are visible, manually adjust the plate position to the region of choice and use the image acquisition software to set the laser power and exposure, image acquisition parameters, and experiment duration period. Select the appropriate GFP and mCherry filters and acquire pulsed transmitted light and fluorescence images every 10 minutes for up to 16 hours, to obtain time-lapse images of the mitosis process.Nine hours after release from the double thymidine block, acquire the images at 12 one-micrometer separated z-planes. Then analyze the images by tracking individual mitotic cells and use the appropriate source software to assemble a corresponding movie. Although most of the control and Cdt1 siRNA cells are at the metaphase stage at nine hours post-release from double thymidine block, fixation at 10 hours reveals that most of the Cdt1 siRNA-treated cells are still arrested in late prometaphase, while the control cells enter anaphase and segregate their chromosomes as expected.Cold treatment nine hours after release from double thymidine block facilitates the retention of stable kinetochore microtubules which are relatively less robust in Cdt1-depleted cells compared to those within control-depleted cells. DNA replication origin licensing is not perturbed in Cdt1-or control-depleted cells during the G2-M phase, as these cells were not found to induce the accumulation of phosphoralated gamma H2AX, a marker for DNA damage, during the subsequent G2 phase. Cdt1 siRNA transfection of asynchronous cultures, however, induces the accumulation of phospho gamma H2AX positive foci possibly due to the DNA damage induced by improper DNA replication licensing.After nuclear envelope breakdown, normal cells enter mitosis and undergo anaphase onset to exit from mitosis within about 30 to 60 minutes. RNAi-mediated knockdown of Hec1, a key kinetochore protein required for robust kinetochore microtubule attachment formation, however, delays the normal mitotic progression to anaphase onset or exit from mitosis even after several hours of mitosis delay. Once mastered, this technique can be completed in three to four days if it is performed properly.While attempting the procedure, it's important to remember to dissolve the thymidine completely after you thaw it out from the freezer. Following this procedure, other methods like western blotting can be performed to answer additional questions like, what are the expression patterns of proteins of interest during mitosis compared to interphase? After its development, this technique paved the way for researchers in the field of cell biology to explore cancer biogenesis in human cell culture models, and to use high-resolution confocal microscopy to identify the cell cycle related functions of proteins of interest.Don't forget that working with reagents like paraformaldehyde and DAPI can be extremely hazardous and that precautions such as wearing gloves and a lab coat, should always be taken while performing this procedure.

combining-mitotic-cell-synchronization-high-resolution-confocal

Research

JoVE Journal

Biology

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles1; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr2. In contrast, cell divisions during plant development are slow, typically on the order of a day 3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).
This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR6, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

Combining Magnetic Sorting of Mother Cells and Fluctuation Tests to Analyze Genome Instability During Mitotic Cell Aging in Saccharomyces cerevisiae

Saccharomyces cerevisiae has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this yeast model is relevant to many organisms, including humans, since DNA repair and DNA damage response factors are well conserved across diverse species. However, S. cerevisiae has not yet been used to fully address whether the rate of accumulating mutations changes with increasing replicative (mitotic) age due to technical constraints. For instance, measurements of yeast replicative lifespan through micromanipulation involve very small populations of cells, which prohibit detection of rare mutations. Genetic methods to enrich for mother cells in populations by inducing death of daughter cells have been developed, but population sizes are still limited by the frequency with which random mutations that compromise the selection systems occur. The current protocol takes advantage of magnetic sorting of surface-labeled yeast mother cells to obtain large enough populations of aging mother cells to quantify rare mutations through phenotypic selections. Mutation rates, measured through fluctuation tests, and mutation frequencies are first established for young cells and used to predict the frequency of mutations in mother cells of various replicative ages. Mutation frequencies are then determined for sorted mother cells, and the age of the mother cells is determined using flow cytometry by staining with a fluorescent reagent that detects bud scars formed on their cell surfaces during cell division. Comparison of predicted mutation frequencies based on the number of cell divisions to the frequencies experimentally observed for mother cells of a given replicative age can then identify whether there are age-related changes in the rate of accumulating mutations. Variations of this basic protocol provide the means to investigate the influence of alterations in specific gene functions or specific environmental conditions on mutation accumulation to address mechanisms underlying genome instability during replicative aging.

Combining Magnetic Sorting of Mother Cells and Fluctuation Tests to Analyze Genome Instability During Mitotic Cell Aging in Saccharomyces cerevisiae

Mutation rates in young Saccharomyces cerevisiae cells measured through fluctuation tests are used to predict mutation frequencies for mother cells of different replicative ages. Magnetic sorting and flow cytometry are then used to measure actual mutation frequencies and age of mother cells to identify any deviations from predicted mutation frequencies.

Saccharomyces cerevisiae has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this ...

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

The cell cycle is important for growth, genome replication, and development in all cells. In bacteria, studies of the cell cycle have focused largely on unsynchronized cells making it difficult to order the temporal events required for cell cycle progression, genome replication, and division. Caulobacter crescentus provides an excellent model system for the bacterial cell cycle whereby cells can be rapidly synchronized in a G0 state by density centrifugation. Cell cycle synchronization experiments have been used to establish the molecular events governing chromosome replication and segregation, to map a genetic regulatory network controlling cell cycle progression, and to identify the establishment of polar signaling complexes required for asymmetric cell division. Here we provide a detailed protocol for the rapid synchronization of Caulobacter NA1000 cells. Synchronization can be performed in a large-scale format for gene expression profiling and western blot assays, as well as a small-scale format for microscopy or FACS assays. The rapid synchronizability and high cell yields of Caulobacter make this organism a powerful model system for studies of the bacterial cell cycle.

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

Synchronization of bacterial cells is essential for studies of the bacterial cell cycle and development. Caulobacter crescentus is synchronizable through density centrifugation allowing a rapid and powerful tool for studies of the bacterial cell cycle. Here we provide a detailed protocol for the synchronization of Caulobacter cells.

The cell cycle is important for growth, genome replication, and development in all cells.  In bacteria, studies of the cell cycle have focused largely on ...

Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell synchronization can be difficult for many cell systems. Standard practice is to block cell cycle progression at a specific stage and then release the accumulated cells producing a wave of cells progressing through the cycle in unison. However, some cell types find this block toxic resulting in abnormal cell cycling, or even mass death. Bromodeoxyuridine (BrdU) uptake can be used to track the cell cycle stage of individual cells. Cells incorporate this synthetic thymidine analog, while synthesizing new DNA during S phase. By providing BrdU for a brief period it is possible to mark a pool of cells that were in S phase while the BrdU was present. These cells can then be tracked through the remainder of the cell cycle and into the next round of replication, permitting the duration of the cell cycle phases to be determined without the need to induce a potentially toxic cell cycle block. It is also possible to determine and correlate the expression of both internal and external proteins during subsequent stages of the cell cycle. These can be used to further refine the assignment of cell cycle stage or assess effects on other cellular functions such as checkpoint activation or cell death.

This protocol describes the use of bromodeoxyuridine (BrdU) uptake to permit the temporal tracking of cells that were in S phase at a specific point in time. Addition of DNA dyes and antibody labeling facilitates detailed analysis of the fate of the S phase cells at later times.

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell ...

A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod shaped chromosomes which are moved individually by the mitotic spindle apparatus. Mitotic chromatin condensation yields chromosomes compacted fifty-fold denser as in interphase. During exit from mitosis, chromosomes have to re-establish their functional interphase state, which is enclosed by a nuclear envelope and is competent for replication and transcription. The decondensation process is morphologically well described, but in molecular terms poorly understood: We lack knowledge about the underlying molecular events and to a large extent the factors involved as well as their regulation. We describe here a cell-free system that faithfully recapitulates chromatin decondensation in vitro, based on mitotic chromatin clusters purified from synchronized HeLa cells and X. laevis egg extract. Our cell-free system provides an important tool for further molecular characterization of chromatin decondensation and its co-ordination with processes simultaneously occurring during mitotic exit such as nuclear envelope and pore complex re-assembly.

The molecular mechanisms of the decondensation of highly compacted mitotic chromatin are ill-defined. We present a cell-free assay based on mitotic chromatin clusters isolated from HeLa cells and Xenopus laevis egg extract that faithfully reconstitutes the decondensation process in vitro.

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod ...

The Microscopy-Based Assay to Study and Analyze the Recycling Endosomes using SNARE Trafficking

Recycling endosomes (REs) are tubular-vesicular organelles generated from early/sorting endosomes in all cell types. These organelles play a key role in the biogenesis of melanosomes, a lysosome-related organelle produced by melanocytes. REs deliver the melanocyte-specific cargo to premature melanosomes during their formation. Blockage in the generation of REs, observed in several mutants of Hermansky-Pudlak syndrome, results in hypopigmentation of skin, hair, and eye. Therefore, studying the dynamics (refer to number and length) of REs is useful to understand the function of these organelles in normal and disease conditions. In this study, we aim to measure the RE dynamics using a resident SNARE STX13.

Recycling endosomes are part of the endosomal tubular network. Here we present a method to quantify the dynamics of recycling endosomes using GFP-STX13 as an organelle marker.

Recycling endosomes (REs) are tubular-vesicular organelles generated from early/sorting endosomes in all cell types. These organelles play a key role in ...

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 ...

Label-Retention Expansion Microscopy (LR-ExM) Enables Super-Resolution Imaging and High-Efficiency Labeling

Expansion microscopy (ExM) is a sample preparation technique that can be combined with most light microscopy methods to increase the resolution. After embedding cells or tissues in swellable hydrogel, samples can be physically expanded three-to sixteen-fold (linear dimension) compared to the original size. Therefore, the effective resolution of any microscope is increased by the expansion factor. A major limitation of the previously introduced ExM is reduced fluorescence after polymerization and the digestion procedure. To overcome this limitation, label-retention expansion microscopy (LR-ExM) has been developed, which prevents signal loss and greatly enhances labeling efficiency using a set of novel trifunctional anchors. This technique allows one to achieve higher resolution when investigating cellular or subcellular structures at a nanometric scale with minimal fluorescent signal loss. LR-ExM can be used not only for immunofluorescence labeling, but also with self-labeling protein tags, such as SNAP- and CLIP-tags, thus achieving higher labeling efficiency. This work presents the procedure and troubleshooting for this immunostaining-based approach, as well as discussion of self-labeling tagging approaches of LR-ExM as an alternative.

Label-Retention Expansion Microscopy LR-ExM Enables Super-Resolution Imaging and High-Efficiency Labeling

A protocol of label retention expansion microscopy (LR-ExM) is demonstrated. LR-ExM uses a novel set of trifunctional anchors, which provides better labeling efficiency compared to previously introduced expansion microscopies.

Expansion microscopy (ExM) is a sample preparation technique that can be combined with most light microscopy methods to increase the resolution. After ...