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Quantitative analysis of actin filament assembly in yeast and plant by live cell fluorescence microscopy
Accepted Manuscript Title: Quantitative analysis of actin filament assembly in yeast and plant by live cell fluorescence microscopy Authors: Joseph Jun Dao Tan, Zhiming Ma, Ying Xie, Liang Yang, Yansong Miao PII: DOI: Reference:
S0968-4328(17)30313-X https://doi.org/10.1016/j.micron.2017.09.011 JMIC 2482
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Please cite this article as: Tan, Joseph Jun Dao, Ma, Zhiming, Xie, Ying, Yang, Liang, Miao, Yansong, Quantitative analysis of actin filament assembly in yeast and plant by live cell fluorescence microscopy.Micron https://doi.org/10.1016/j.micron.2017.09.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title: Quantitative analysis of actin filament assembly in yeast and plant by live cell fluorescence microscopy Joseph Jun Dao Tan1,3, Zhiming Ma1, Ying Xie2, Liang Yang1,4, and Yansong Miao1,2 1
School of Biological Sciences, Nanyang Technological University, Singapore 637551, Singapore. School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore 637459, Singapore. 3 Ageing Research Institute for Society and Education, Interdisciplinary Graduate School, Nanyang Technological University, Singapore 639798, Singapore. 4 Singapore Centre for Environmental Life Sciences Engineering (SCELSE), Nanyang Technological University, Singapore 637551, Singapore. 2
ABSTRACT Eukaryotic cells depend on a dynamic actin cytoskeleton to regulate many conserved intracellular events such as endocytosis, morphogenesis, polarized cell growth, and cytokinesis (Engqvist-Goldstein & Drubin, 2003, Salbreux et al., 2012, Pruyne et al., 2004, Pollard, 2010). These activities depend on a precise and well-organized spatiotemporal actin assembly that involves many conserved processes found in eukaryotic cells ranging from a unicellular organism, such as yeast, to multicellular organisms, such as plants and human. In particular, both budding yeast Saccharomyces cerevisiae and plant Arabidopsis thaliana have been proven to be the powerful and great model organisms to study the molecular mechanisms of the polymerization of the actin cytoskeleton and the actin-driven processes in walled-cells. Here we describe the methods in imaging and image processing to analyze dynamic actin filament assembly in budding yeast and Arabidopsis using a wide-field fluorescent microscope. 1. Introduction In response to physiological or pathological signals, cell rapidly changes their morphology and cellular processes, which depend largely on the dynamic assembly and disassembly of actin cytoskeleton networks. Cells have multiple mechanisms to initiate new actin filaments, such as using actin-related protein (Arp)2/3 complex and formins (Pollard, 2007). While Arp2/3 complex anchors the pre-existing filaments to generate new branching filamentous actin, formins generate unbranched actin filaments and actin bundles for vesicular delivery, polarized cell growth, and the assembly of cytokinetic contraction ring (Chesarone et al., 2010). Although the essential functions of the Arp2/3 complex for endocytosis and cell migration are well documented in both yeast and animals (Pollard & Cooper, 2009, Engqvist-Goldstein & Drubin, 2003, Pollard, 2007), the importance of Arp2/3 in plants is still unclear because the mutants of Arp2/3 complex components only have minor cell biological and developmental defects (Li et al., 2003). Plants have more than 20 formins that have been found to be involved in multiple plant developmental process, but only a few plant formins have been studied with clear functionality (Ingouff et al., 2005, Michelot et al., 2005, Cheung et al., 2010). The mechanistic studies of actin filament polymerization and depolymerization have been facilitated by living cell fluorescent imaging approaches. Fluorescent living cell materials that study actin cytoskeleton and actin-binding proteins were usually generated through homology recombination-based gene editing in yeast or TDNA insertion-based transformation in the plant. High-quality live cell imaging and quantitative measurement of fluorescent signals are both critical in studying the dynamic behaviors of actin filament assembly and the actin assembly driven-biological
processes. In budding yeast, the force perpendicular to the membrane was generated by Arp2/3nucleated actin networks that drive the membrane bending and the endocytic invagination (Kaksonen et al., 2003, Kaksonen et al., 2005). After scission, released endosomes are transported along the tracks of actin cables that are nucleated by formins. (Miao et al., 2013, Toshima et al., 2006). Both the branched and unbranched actin filaments are stabilized by actin-binding proteins, such as actin crosslinking proteins and tropomyosin (Miao et al., 2016, Xie et al., 2017). Quantitative imaging and image analysis of actin filaments would differentiate the subtle changes of actin assembly, including the bundling and morphology of actin filaments (Miao et al., 2016, Miao et al., 2013). In plant research, imaging of dynamic actin filaments assembly reveals multiple intracellular activities and polarized plant growth, including root hair and pollen tube growth (Liu et al., 2015). Lifeact is a 17 amino acid peptide derived from the N-terminus of yeast Abp140 protein that decorates F-actin with minimum perturbation of actin dynamics when expressed at optimized lower expression levels (Era et al., 2009). In addition, a GFP fusion of the actin-binding domain of the Arabidopsis fimbrin1 (AtFIM1) protein (GFP-fABD2) is also widely used without obvious adverse effects on plant development (Henty-Ridilla et al., 2013, Cao et al., 2016). By following the actin dynamics in Arabidopsis pollen tube, the functional interactions between profilin and formin modules are well deciphered for actin filament polymerization and pollen tube elongation (Liu et al., 2015). Besides the physiological importance of actin assembly in plant development and polarized cell growth, actin filaments in eukaryotic cells have also been well known to be targets of various virulence factors of bacterial pathogens (Henty-Ridilla et al., 2013, Kang et al., 2014a). The changes of actin cytoskeleton assembly were demonstrated during plant innate immune responses or effector-triggered responses. Several molecular patterns and type III secretion system produced effectors from bacterial pathogens are known to modulate host actin filament assembly during defense responses. Upon pathogen attack, host actin assembly and actin network organization are changed by the identification of flagellum association (Henty-Ridilla et al., 2013). For example, actin-targeting effector protein HopW1 produced by Pseudomonas syringae could also disrupt actin-dependent endocytosis when expressed in plant cells (Kang et al., 2014b). Here we focus on describing the workflows of sample preparation, image acquisition, and image analysis in both the budding yeast and Arabidopsis, using conventional epifluorescence microscopy and data processing with ImageJ and Imaris. In budding yeast, GFP fused Abp140 and RFP fused Abp1 were used as markers for branched actin patches and unbranched actin cables, respectively. To visualize and quantify actin filament in Arabidopsis, transgenic Arabidopsis of Lifeact-Venus was used under the control of cauliflower mosaic virus 35S (35S) promoter (Era et al., 2009). Root hair is a great cell model to study the polarized cell growth in plants. We shall describe the imaging of root hair actin filaments by conventional wide-field microscope. 2. Materials for budding yeast imaging 2.1
Yeast strains The following yeast strain used in the review was constructed by tagging Abp1 and Abp140 with red fluorescent proteins (RFP) and 3× green fluorescent proteins (3GFP) at their individual Cterminal respectively using the homologous recombinant method. Name YM_Y753
2.2
Culture medium
Genotype MATa his3-∆200 leu2-3, 112 ura3-52 ABP1-RFP::HIS3 ABP1403GFP::HIS3
Yeast extract Peptone Dextrose (YPD) plates: 2% bacto-peptone, 1% bacto yeast extract, 2% bactoagar and 2% D-Glucose. Synthetic drop-out (SD) tryptophan: 0.067% yeast nitrogen base, 20 g/mL for each amino acid of adenine, histidine, L-leucine, uracil, and 2% D-glucose. 2.3
Chemicals Concanavalin A (ConA): Canavalia ensiformis (Jack bean) Type IV, lyophilized powder (Sigma, USA). Prepare a stock solution of 10 mg/mL by dissolving the lyophilized powder in double distilled water. Aliquot and keep in -20°C. Prepare a working concentration of 1 mg/mL in double distilled water prior to cell imaging.
2.4
Image acquisition Live-cell images were taken by fluorescence microscopy Leica DMi8 (Leica, Germany) that is equipped with a 100X objective lens (HCX PL APO 100×, N.A 1.4), SPECTRA X light engine (Lumencor, USA), and a sCMOS camera ORCA-Flash4.0 LT Digital (Hamamatsu, Japan). Immersion oil used is Type F Immersion liquid, ISO 8036 (Leica Microsystems, Germany). GFPtagged constructs were excited at 485/25 nm wavelength, and fluorescence was detected at 525/50 nm wavelength. Images were acquired using the MetaMorph software (Molecular Devices). Yeast cells were stabilized by ConA using the imaging chamber (Attofluor Cell Chamber, Thermo Fisher Scientific).
3. Step by step imaging protocol for cell imaging 3.1
Under room temperature 1. Recover the yeast strains from -80°C freezer on a fresh YPD plate at 30°C. The day after, streak one single colony on the YPD plate into 5 ml of SD tryptophan and inoculate overnight at 25°C. 2. Re-inoculate the cell culture in SD tryptophan at a starting OD600 = 0.2 to OD600 = 0.5-0.7. 3. Coat the coverslip with 50 L of ConA (1 mg/mL) for 10 mins (Fig. 1C). Wash the ConA away with 100 μL double distilled water. 4. Add 500 μL of cell culture and incubate for 10 mins at room temperature (Fig. 1D). 5. Apply an adequate amount of immersion oil on the objective lens and mount the metal chamber. 6. Level the focus plane to the middle focal plane of yeast cells to ensure that average direction of the endocytic vesicle is in parallel or lies on the focus plane. 7. Input the parameters for image acquisition. For acquiring z-stacks of Abp1-RFP and Abp1403GFP, a laser power of 200 and exposure time of 150 ms was used. When using a different wide-field microscope with the different light source, we follow a general guideline to decide. We tried to minimize the photo-bleaching by strong LED light but retain sufficient fluorescent signal to ensure the image quality for quantitative analysis. Optimizations of image conditions are required for fluorescent proteins with different signal intensity and dynamics. 8. For taking time-lapse movies of Abp1-RFP, level the focal plane to the middle focal plane of cells. Use a laser power of 50 and exposure time of 1s. 9. To take time-lapse movies of Abp140-3GFP, level the focal plane to the cortical plane of the cells. The laser power is 40 and exposure time is 250ms.
3.2
Under heat stress
10. For imaging under heat stress, switch on the heat chamber 15 mins prior to the start of the experiment to allow for thermal equilibrium. To achieve accurate temperature control, a PIDcontrolled close-loop heater equipped with Pt100 temperature sensor was used. 11. Follow steps 3-9 for imaging. 4. Quantitative imaging analysis of actin patch lifetime 4.1
Image processing Z-stack images and movies were deconvoluted using Huygens Deconvolution software package (Scientific Volume Imaging, Netherlands). The following parameters were used: iterations = 50, signal to noise = 40, quality threshold = 0.01. The lifetime analysis of Abp1-RFP was done using ImageJ (http://imagej.nih.gov/ij/).
4.2
Measuring actin patch lifetime 1. Open the deconvoluted time-lapse movie of Abp1-RFP in Image J. 2. Generate the kymograph (Fig. 2C) using the “Reslice” function in “Stacks” of Image J. 3. Calculate the lifetime by measuring the distance between the appearance and disappearance of the fluorescent track, depending on the exposure time and time interval during the acquisition.
Fig. 1 Experimental setup. (A) Side view of the metal chamber. Assemble the top and bottom of chamber with the coverslip in between. (B) Top view of the metal chamber. (C) Coat the coverslip with ConA (1 mg/mL) for 10 mins and wash away with double distilled water. (D) Add 500 L-1 mL of cell culture and wait for 10 mins.
Fig. 2 Actin dynamics in budding yeast. (A) Maximum z-projections of actin cables and patches at heat stress condition. Cells were incubated at the indicated temperature for 20 mins before imaging. The red signal represents Abp1-RFP, the green signal represents Abp140-3GFP. Images are shown after deconvolution. (B) A simplified model of tracking actin assembly during endocytosis using a lifetime of Abp1-RFP. (C) Kymographs show the lifetime of Abp1-RFP under different heat stress (D) Quantification of the lifetime of Abp1-RFP, n=100 patches; ***P < 0.001. Error bar indicates SD.
5. Materials for Arabidopsis imaging 5.1
Samples preparation Lifeact-Venus transgenic Arabidopsis thaliana was used to visualize actin cytoskeleton in root hair (Era et al., 2009). Arabidopsis seeds were surface-sterilized in 15% bleach (v/v) for 3 mins. After washing three times with sterilized distilled water, the seeds were stratified at 4°C in the dark for at least 2 days, and were then grown vertically on 1/2 × Murashige and Skoog (MS) medium solidified by 0.8% agar at pH 5.8. The seedlings were grown in a growth chamber (Panasonic MLR-352H-PE) with photoperiodic lighting (16 hours of light and 8 hours of dark) at 22°C with a relative humidity of 75%. 6-7 days’ seedlings were used for imaging.
5.2
Image acquisition The microscope used is similar to section 2.4
6. Imaging and quantitative analysis of actin filament in Arabidopsis root hair 6.1
Imaging The Arabidopsis roots were mounted in a slice and then covered with a coverslip. The LifeactVenus signal was excited at 485/25 nm wavelength, and fluorescence was detected at 525/50 nm wavelength. Time-lapse imaging was applied to record the actin filament organization and dynamic in Arabidopsis root hair. The focal plane determined at a potision where most of the actin filaments in the root hair are in good focus. A laser power of 10 and exposure time of 250 ms was used, 5s interval. At least 20 frames were recorded in each time-lapse image.
6.2
Quantify actin filament dynamics 1. Open the time-lapse images in ImageJ. 2. Choose the images taken at time points 0s, 30s, 60s from the stack using the ‘Duplicate’ function of ImageJ. 3. Label the signal in these three images with blue, green and red colors (Fig. 3A) using the ‘Lookup Tables’ of ImageJ. 4. Merge the three images using the ‘Color → Merge channel’ function. The overlapped signal from blue, green and red filaments generate a white signal that indicates low actin dynamic. 5. To further analyze the actin filament dynamic in details. Three straight lines were selected in different root hair regions (0-30m, 30-60m, 60- 90m from the root tip) as ROI (Region Of Interests) (Fig. 3A). For each ROI, signal intensity histogram was generated in each channel using ‘Analyze →Plot Profile’ functions in ImageJ. Overlapped signal peaks indicate low actin dynamic over image acquisition(Fig. 3B-D).
6.3
Quantify actin filament mean intensity 1. Choose the first frame of the time-lapse images and open the images in Imaris software (Bitplane, USA). 2. Select ‘Add new Filament’, and then ‘Edit Manually’. 3. Select a filament starting point on the image and press ‘shift + right click’. A presumptive line will be generated by moving the mouse cursor along the filament. Only these well-focused filaments with sharp and clear edge were traced and quantified (Fig. 3E). 4. Select all the traced filaments and adjust the threshold until all the presumptive lines can match with the original filaments. 5. Analyze the traced filaments by applying ‘analyze’ function of Imaris. The output data were analyzed statistically using GraphPad Prism software (GraphPad, USA).
Fig. 3 The organization and dynamic of the actin filaments (LifeAct-Venus) in Arabidopsis root hair. (A) Actin filaments indicated by Lifeact-Venus in growing root hair were shown with 10s intervals between each acquisition. Three frames with 30s interval were labeled with blue (0s), green (30s) and red (60s) colours, which were merged in the last row to demonstrate the filament dynamics over time. The overlapped signal from blue, green and red filaments generate a white signal that indicates the low actin dynamic over the imaging period. (B-D) One representative signal histograms generated from three ROI in (A), which are indicated by the arrows in different root hair regions (0-30 m, 30-60 m, 60-90 m away from the tip). (E) An example of actin filament signal tracking in different root hair regions done using Imaris. (F) Mean intensity of actin filaments in three different root hair regions (n=29, n=27, and n=22 from left to right). Pvalues were determined by two-tailed Student’s t-test assuming equal variances (****p < 0.0001). AU, arbitrary unit. Error bars indicate SD. Scale bar, 5m.
7. Discussion 7.1
Actin assembly and disassembly in yeast and plant root hair In budding yeast, actin plays a major role in endocytosis, especially on membrane invagination. A functional actin network with active filament turnover is essential for driving the endocytosis (Kaksonen et al., 2003). Many actin-binding proteins localize at cortical patches to facilitate the actin assembly and thus the endocytosis (Fig. 2B). Actin filaments are nucleated by Arp2/3
complex or formin proteins, which is followed by a competitive and coordinated function of multiple actin-binding proteins, including the elongation, crossing linking proteins and depolymerization factors (Lappalainen & Drubin, 1997, Rodal et al., 1999, Chen et al., 2015, Michelot & Drubin, 2011, Okreglak & Drubin, 2010). Due to the ability in binding to the branched actin filament, we can use the fluorescent signal of Abp1-RFP imaged by fluorescent wide-field microscopy to trace the assembly and disassembly of actin filaments during endocytosis from each cortical patch (Fig. 2C). On average, the lifetime of actin patches labelled by Abp1-RFP is 10s. In such short window of time, assembled actin network generates sufficient mechanical force to facilitate the internalization of endocytic pits from the plasma membrane. Upon a heat stress treatment, a general increase of endocytosis efficiency will be observed, in which the lifetime of actin patches labelled by Abp1-RFP decreases to 8s on average (Fig. 2D). Heat stress also affected the polarized localization of endocytic sites in the cell. Under room temperature, the cortical patches are largely concentrated in the bud, but when the temperature increases to 39°C within 30 minutes, the actin patches were depolarized and showed more endocytic sites in the mother cell around the cell periphery (Fig. 2A). Actin patches will relocate back to the bud after 30 min despite the persistence of the heat treatment, which is a depolarization and repolarization phenomena for polarity proteins and actin cytoskeleton that are involved in multiple signaling pathways (Levin, 2011, Guo et al., 2009). To compare the actin cytoskeleton at the polarized and depolarized stages, we imaged the cells at 20 min heat stress condition. Under heat stress, actin patches are depolarized, and actin cables are less defined and undergo an architectural reorganization, suggesting the changes in coordinated actin filament assembly. The Arabidopsis root hair serves as an ideal model to study actin cytoskeleton regulation in a polarized plant cell. The fast polarized cell growth highly depends on the polarized organization of cytoplasmic actin networks (Carol & Dolan, 2002). Dynamic actin filaments assembly and disassembly are critical for supporting fast polarized cell growth of plant root hair. Actin filament bundles create stable networks for vesicular trafficking and generating mechanical forces for polarized cell growth. Interestingly, actin filament in different positions of polarized root hair cells shows different dynamic behaviors. Those actin filaments in the base region are relatively more stable with a higher level of actin bundles, revealed by the quantitative measurement of actin filament diameter and mean intensity (Fig. 3C and 3D). Due to the limitation in resolution of widefield microscopy, actin filament diameter is less informative in general to indicate the precise actin bundle structure. Therefore, an increase of filament diameter from the tip to base root hair region does not represent a linear increase of actin filament number in each bundle, though a relative modulation of bundling level is effectively presented. Such difference between different zones of root hair could be explained by the cellular content at each region. In the root hair base region, most of the cytoplasm is occupied by a central vacuole, and no cell expansion happens in the region (Ketelaar, 2013). Plant protein sorting and vesicular delivery are involved in multiple intracellular activities of membrane transport and cell growth (Miao et al., 2011, Miao & Jiang, 2007, Miao et al., 2008, Miao et al., 2006, Wang et al., 2009, Kim et al., 2010, Min et al., 2007). The highly bundled actin cytoskeleton serves as a back bone to maintain the cell shape and is involved in the long-distance delivering of vesicles to the root hair tip. However, with the extension to tips, the actin filaments disperse to thinner and more dynamic filaments gradually. In the fast-growing tip, the thinner and dynamic actin filaments allow the fast exchange of membranes between growing tip and intracellular organelles (Pei et al., 2012). Such different actin dynamics at different cell positions may also suggest different stabilization in actin network formation, which worth of future studies in understanding the mechanisms by which the actin network assembly are well-cooperated
by actin-binding proteins during polarized root hair growth. However, using Lifeact as actin filament marker require careful design and caution in results interpretation. mCherry fluorescent protein fused Liftact was indicated to have potential perturbation in actin filament polymerization in a concentration-dependent manner (Courtemanche et al., 2016). An appropriate selection of fluorescent probe and the control of fluorescent protein expression level should be taken into consideration in experimental design. 7.2
Technique considerations for imaging and image analysis 7.2.1
Improving image resolution and contrast during acquisition
Most budding yeast strains are auxotrophic and the growth media needs to be supplemented with essential amino acids. However, some amino acid can hinder and increase the background fluorescence such as tryptophan. Hence it is advised to do imaging using growth media without tryptophan. In addition, to achieve high image resolution and quality, we use immersion oil with a refractive index of n= 1.518 to match the refractive index of the imaging medium and cover glass (thickness, 0.17 mm). 7.2.2
Consideration of using deconvolution to improve image quality
Raw images collected by the epifluorescence microscope can give high background noise caused by blurring from diffraction and photon noise, especially when imaging samples with dim signals. Applying deconvolution can help to enhance the signal to noise ratio by removing unwanted background while improving the signal of interest. This works the best for analyzing lifetime of actin patches as it improves the contrast of kymographs of the lifetime of yeast endocytic proteins before data analysis. However, not all images are suitable for deconvolution. Original images with high background noise or weak fluorescence signal are not advised for deconvolution. Only apply deconvolution to improve the resolution and contrast while not compromising the integrity of images. Below are some tips to consider before deconvolution: I. Remove unwanted slices in a z-stacks that are often out of plane or focus on improving the deconvolution results; II. For budding yeast analysis, crop out regions of interest that has better signal and disregard areas with larger background noise can improve the final resolution. 8. Acknowledgements This work was supported by grants from Nanyang Technological University NAP start-up grant, NIMBELS (NIM/01/2016), and MOE Tier 2 (MOE2016-T2-1-005) to Y. MIAO. 9. References Cao L, Henty‐Ridilla JL, Blanchoin L, Staiger CJ, 2016. Profilin‐Dependent Nucleation and Assembly of Actin Filaments Controls Cell Elongation in Arabidopsis. Plant Physiol 170, 220‐33. Carol RJ, Dolan L, 2002. Building a hair: tip growth in Arabidopsis thaliana root hairs. Philosophical Transactions of the Royal Society of London B: Biological Sciences 357, 815‐21.
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S0968-4328(17)30313-X https://doi.org/10.1016/j.micron.2017.09.011 JMIC 2482
To appear in:
Micron
Received date: Revised date: Accepted date:
15-8-2017 25-9-2017 25-9-2017
Please cite this article as: Tan, Joseph Jun Dao, Ma, Zhiming, Xie, Ying, Yang, Liang, Miao, Yansong, Quantitative analysis of actin filament assembly in yeast and plant by live cell fluorescence microscopy.Micron https://doi.org/10.1016/j.micron.2017.09.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title: Quantitative analysis of actin filament assembly in yeast and plant by live cell fluorescence microscopy Joseph Jun Dao Tan1,3, Zhiming Ma1, Ying Xie2, Liang Yang1,4, and Yansong Miao1,2 1
School of Biological Sciences, Nanyang Technological University, Singapore 637551, Singapore. School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore 637459, Singapore. 3 Ageing Research Institute for Society and Education, Interdisciplinary Graduate School, Nanyang Technological University, Singapore 639798, Singapore. 4 Singapore Centre for Environmental Life Sciences Engineering (SCELSE), Nanyang Technological University, Singapore 637551, Singapore. 2
ABSTRACT Eukaryotic cells depend on a dynamic actin cytoskeleton to regulate many conserved intracellular events such as endocytosis, morphogenesis, polarized cell growth, and cytokinesis (Engqvist-Goldstein & Drubin, 2003, Salbreux et al., 2012, Pruyne et al., 2004, Pollard, 2010). These activities depend on a precise and well-organized spatiotemporal actin assembly that involves many conserved processes found in eukaryotic cells ranging from a unicellular organism, such as yeast, to multicellular organisms, such as plants and human. In particular, both budding yeast Saccharomyces cerevisiae and plant Arabidopsis thaliana have been proven to be the powerful and great model organisms to study the molecular mechanisms of the polymerization of the actin cytoskeleton and the actin-driven processes in walled-cells. Here we describe the methods in imaging and image processing to analyze dynamic actin filament assembly in budding yeast and Arabidopsis using a wide-field fluorescent microscope. 1. Introduction In response to physiological or pathological signals, cell rapidly changes their morphology and cellular processes, which depend largely on the dynamic assembly and disassembly of actin cytoskeleton networks. Cells have multiple mechanisms to initiate new actin filaments, such as using actin-related protein (Arp)2/3 complex and formins (Pollard, 2007). While Arp2/3 complex anchors the pre-existing filaments to generate new branching filamentous actin, formins generate unbranched actin filaments and actin bundles for vesicular delivery, polarized cell growth, and the assembly of cytokinetic contraction ring (Chesarone et al., 2010). Although the essential functions of the Arp2/3 complex for endocytosis and cell migration are well documented in both yeast and animals (Pollard & Cooper, 2009, Engqvist-Goldstein & Drubin, 2003, Pollard, 2007), the importance of Arp2/3 in plants is still unclear because the mutants of Arp2/3 complex components only have minor cell biological and developmental defects (Li et al., 2003). Plants have more than 20 formins that have been found to be involved in multiple plant developmental process, but only a few plant formins have been studied with clear functionality (Ingouff et al., 2005, Michelot et al., 2005, Cheung et al., 2010). The mechanistic studies of actin filament polymerization and depolymerization have been facilitated by living cell fluorescent imaging approaches. Fluorescent living cell materials that study actin cytoskeleton and actin-binding proteins were usually generated through homology recombination-based gene editing in yeast or TDNA insertion-based transformation in the plant. High-quality live cell imaging and quantitative measurement of fluorescent signals are both critical in studying the dynamic behaviors of actin filament assembly and the actin assembly driven-biological
processes. In budding yeast, the force perpendicular to the membrane was generated by Arp2/3nucleated actin networks that drive the membrane bending and the endocytic invagination (Kaksonen et al., 2003, Kaksonen et al., 2005). After scission, released endosomes are transported along the tracks of actin cables that are nucleated by formins. (Miao et al., 2013, Toshima et al., 2006). Both the branched and unbranched actin filaments are stabilized by actin-binding proteins, such as actin crosslinking proteins and tropomyosin (Miao et al., 2016, Xie et al., 2017). Quantitative imaging and image analysis of actin filaments would differentiate the subtle changes of actin assembly, including the bundling and morphology of actin filaments (Miao et al., 2016, Miao et al., 2013). In plant research, imaging of dynamic actin filaments assembly reveals multiple intracellular activities and polarized plant growth, including root hair and pollen tube growth (Liu et al., 2015). Lifeact is a 17 amino acid peptide derived from the N-terminus of yeast Abp140 protein that decorates F-actin with minimum perturbation of actin dynamics when expressed at optimized lower expression levels (Era et al., 2009). In addition, a GFP fusion of the actin-binding domain of the Arabidopsis fimbrin1 (AtFIM1) protein (GFP-fABD2) is also widely used without obvious adverse effects on plant development (Henty-Ridilla et al., 2013, Cao et al., 2016). By following the actin dynamics in Arabidopsis pollen tube, the functional interactions between profilin and formin modules are well deciphered for actin filament polymerization and pollen tube elongation (Liu et al., 2015). Besides the physiological importance of actin assembly in plant development and polarized cell growth, actin filaments in eukaryotic cells have also been well known to be targets of various virulence factors of bacterial pathogens (Henty-Ridilla et al., 2013, Kang et al., 2014a). The changes of actin cytoskeleton assembly were demonstrated during plant innate immune responses or effector-triggered responses. Several molecular patterns and type III secretion system produced effectors from bacterial pathogens are known to modulate host actin filament assembly during defense responses. Upon pathogen attack, host actin assembly and actin network organization are changed by the identification of flagellum association (Henty-Ridilla et al., 2013). For example, actin-targeting effector protein HopW1 produced by Pseudomonas syringae could also disrupt actin-dependent endocytosis when expressed in plant cells (Kang et al., 2014b). Here we focus on describing the workflows of sample preparation, image acquisition, and image analysis in both the budding yeast and Arabidopsis, using conventional epifluorescence microscopy and data processing with ImageJ and Imaris. In budding yeast, GFP fused Abp140 and RFP fused Abp1 were used as markers for branched actin patches and unbranched actin cables, respectively. To visualize and quantify actin filament in Arabidopsis, transgenic Arabidopsis of Lifeact-Venus was used under the control of cauliflower mosaic virus 35S (35S) promoter (Era et al., 2009). Root hair is a great cell model to study the polarized cell growth in plants. We shall describe the imaging of root hair actin filaments by conventional wide-field microscope. 2. Materials for budding yeast imaging 2.1
Yeast strains The following yeast strain used in the review was constructed by tagging Abp1 and Abp140 with red fluorescent proteins (RFP) and 3× green fluorescent proteins (3GFP) at their individual Cterminal respectively using the homologous recombinant method. Name YM_Y753
2.2
Culture medium
Genotype MATa his3-∆200 leu2-3, 112 ura3-52 ABP1-RFP::HIS3 ABP1403GFP::HIS3
Yeast extract Peptone Dextrose (YPD) plates: 2% bacto-peptone, 1% bacto yeast extract, 2% bactoagar and 2% D-Glucose. Synthetic drop-out (SD) tryptophan: 0.067% yeast nitrogen base, 20 g/mL for each amino acid of adenine, histidine, L-leucine, uracil, and 2% D-glucose. 2.3
Chemicals Concanavalin A (ConA): Canavalia ensiformis (Jack bean) Type IV, lyophilized powder (Sigma, USA). Prepare a stock solution of 10 mg/mL by dissolving the lyophilized powder in double distilled water. Aliquot and keep in -20°C. Prepare a working concentration of 1 mg/mL in double distilled water prior to cell imaging.
2.4
Image acquisition Live-cell images were taken by fluorescence microscopy Leica DMi8 (Leica, Germany) that is equipped with a 100X objective lens (HCX PL APO 100×, N.A 1.4), SPECTRA X light engine (Lumencor, USA), and a sCMOS camera ORCA-Flash4.0 LT Digital (Hamamatsu, Japan). Immersion oil used is Type F Immersion liquid, ISO 8036 (Leica Microsystems, Germany). GFPtagged constructs were excited at 485/25 nm wavelength, and fluorescence was detected at 525/50 nm wavelength. Images were acquired using the MetaMorph software (Molecular Devices). Yeast cells were stabilized by ConA using the imaging chamber (Attofluor Cell Chamber, Thermo Fisher Scientific).
3. Step by step imaging protocol for cell imaging 3.1
Under room temperature 1. Recover the yeast strains from -80°C freezer on a fresh YPD plate at 30°C. The day after, streak one single colony on the YPD plate into 5 ml of SD tryptophan and inoculate overnight at 25°C. 2. Re-inoculate the cell culture in SD tryptophan at a starting OD600 = 0.2 to OD600 = 0.5-0.7. 3. Coat the coverslip with 50 L of ConA (1 mg/mL) for 10 mins (Fig. 1C). Wash the ConA away with 100 μL double distilled water. 4. Add 500 μL of cell culture and incubate for 10 mins at room temperature (Fig. 1D). 5. Apply an adequate amount of immersion oil on the objective lens and mount the metal chamber. 6. Level the focus plane to the middle focal plane of yeast cells to ensure that average direction of the endocytic vesicle is in parallel or lies on the focus plane. 7. Input the parameters for image acquisition. For acquiring z-stacks of Abp1-RFP and Abp1403GFP, a laser power of 200 and exposure time of 150 ms was used. When using a different wide-field microscope with the different light source, we follow a general guideline to decide. We tried to minimize the photo-bleaching by strong LED light but retain sufficient fluorescent signal to ensure the image quality for quantitative analysis. Optimizations of image conditions are required for fluorescent proteins with different signal intensity and dynamics. 8. For taking time-lapse movies of Abp1-RFP, level the focal plane to the middle focal plane of cells. Use a laser power of 50 and exposure time of 1s. 9. To take time-lapse movies of Abp140-3GFP, level the focal plane to the cortical plane of the cells. The laser power is 40 and exposure time is 250ms.
3.2
Under heat stress
10. For imaging under heat stress, switch on the heat chamber 15 mins prior to the start of the experiment to allow for thermal equilibrium. To achieve accurate temperature control, a PIDcontrolled close-loop heater equipped with Pt100 temperature sensor was used. 11. Follow steps 3-9 for imaging. 4. Quantitative imaging analysis of actin patch lifetime 4.1
Image processing Z-stack images and movies were deconvoluted using Huygens Deconvolution software package (Scientific Volume Imaging, Netherlands). The following parameters were used: iterations = 50, signal to noise = 40, quality threshold = 0.01. The lifetime analysis of Abp1-RFP was done using ImageJ (http://imagej.nih.gov/ij/).
4.2
Measuring actin patch lifetime 1. Open the deconvoluted time-lapse movie of Abp1-RFP in Image J. 2. Generate the kymograph (Fig. 2C) using the “Reslice” function in “Stacks” of Image J. 3. Calculate the lifetime by measuring the distance between the appearance and disappearance of the fluorescent track, depending on the exposure time and time interval during the acquisition.
Fig. 1 Experimental setup. (A) Side view of the metal chamber. Assemble the top and bottom of chamber with the coverslip in between. (B) Top view of the metal chamber. (C) Coat the coverslip with ConA (1 mg/mL) for 10 mins and wash away with double distilled water. (D) Add 500 L-1 mL of cell culture and wait for 10 mins.
Fig. 2 Actin dynamics in budding yeast. (A) Maximum z-projections of actin cables and patches at heat stress condition. Cells were incubated at the indicated temperature for 20 mins before imaging. The red signal represents Abp1-RFP, the green signal represents Abp140-3GFP. Images are shown after deconvolution. (B) A simplified model of tracking actin assembly during endocytosis using a lifetime of Abp1-RFP. (C) Kymographs show the lifetime of Abp1-RFP under different heat stress (D) Quantification of the lifetime of Abp1-RFP, n=100 patches; ***P < 0.001. Error bar indicates SD.
5. Materials for Arabidopsis imaging 5.1
Samples preparation Lifeact-Venus transgenic Arabidopsis thaliana was used to visualize actin cytoskeleton in root hair (Era et al., 2009). Arabidopsis seeds were surface-sterilized in 15% bleach (v/v) for 3 mins. After washing three times with sterilized distilled water, the seeds were stratified at 4°C in the dark for at least 2 days, and were then grown vertically on 1/2 × Murashige and Skoog (MS) medium solidified by 0.8% agar at pH 5.8. The seedlings were grown in a growth chamber (Panasonic MLR-352H-PE) with photoperiodic lighting (16 hours of light and 8 hours of dark) at 22°C with a relative humidity of 75%. 6-7 days’ seedlings were used for imaging.
5.2
Image acquisition The microscope used is similar to section 2.4
6. Imaging and quantitative analysis of actin filament in Arabidopsis root hair 6.1
Imaging The Arabidopsis roots were mounted in a slice and then covered with a coverslip. The LifeactVenus signal was excited at 485/25 nm wavelength, and fluorescence was detected at 525/50 nm wavelength. Time-lapse imaging was applied to record the actin filament organization and dynamic in Arabidopsis root hair. The focal plane determined at a potision where most of the actin filaments in the root hair are in good focus. A laser power of 10 and exposure time of 250 ms was used, 5s interval. At least 20 frames were recorded in each time-lapse image.
6.2
Quantify actin filament dynamics 1. Open the time-lapse images in ImageJ. 2. Choose the images taken at time points 0s, 30s, 60s from the stack using the ‘Duplicate’ function of ImageJ. 3. Label the signal in these three images with blue, green and red colors (Fig. 3A) using the ‘Lookup Tables’ of ImageJ. 4. Merge the three images using the ‘Color → Merge channel’ function. The overlapped signal from blue, green and red filaments generate a white signal that indicates low actin dynamic. 5. To further analyze the actin filament dynamic in details. Three straight lines were selected in different root hair regions (0-30m, 30-60m, 60- 90m from the root tip) as ROI (Region Of Interests) (Fig. 3A). For each ROI, signal intensity histogram was generated in each channel using ‘Analyze →Plot Profile’ functions in ImageJ. Overlapped signal peaks indicate low actin dynamic over image acquisition(Fig. 3B-D).
6.3
Quantify actin filament mean intensity 1. Choose the first frame of the time-lapse images and open the images in Imaris software (Bitplane, USA). 2. Select ‘Add new Filament’, and then ‘Edit Manually’. 3. Select a filament starting point on the image and press ‘shift + right click’. A presumptive line will be generated by moving the mouse cursor along the filament. Only these well-focused filaments with sharp and clear edge were traced and quantified (Fig. 3E). 4. Select all the traced filaments and adjust the threshold until all the presumptive lines can match with the original filaments. 5. Analyze the traced filaments by applying ‘analyze’ function of Imaris. The output data were analyzed statistically using GraphPad Prism software (GraphPad, USA).
Fig. 3 The organization and dynamic of the actin filaments (LifeAct-Venus) in Arabidopsis root hair. (A) Actin filaments indicated by Lifeact-Venus in growing root hair were shown with 10s intervals between each acquisition. Three frames with 30s interval were labeled with blue (0s), green (30s) and red (60s) colours, which were merged in the last row to demonstrate the filament dynamics over time. The overlapped signal from blue, green and red filaments generate a white signal that indicates the low actin dynamic over the imaging period. (B-D) One representative signal histograms generated from three ROI in (A), which are indicated by the arrows in different root hair regions (0-30 m, 30-60 m, 60-90 m away from the tip). (E) An example of actin filament signal tracking in different root hair regions done using Imaris. (F) Mean intensity of actin filaments in three different root hair regions (n=29, n=27, and n=22 from left to right). Pvalues were determined by two-tailed Student’s t-test assuming equal variances (****p < 0.0001). AU, arbitrary unit. Error bars indicate SD. Scale bar, 5m.
7. Discussion 7.1
Actin assembly and disassembly in yeast and plant root hair In budding yeast, actin plays a major role in endocytosis, especially on membrane invagination. A functional actin network with active filament turnover is essential for driving the endocytosis (Kaksonen et al., 2003). Many actin-binding proteins localize at cortical patches to facilitate the actin assembly and thus the endocytosis (Fig. 2B). Actin filaments are nucleated by Arp2/3
complex or formin proteins, which is followed by a competitive and coordinated function of multiple actin-binding proteins, including the elongation, crossing linking proteins and depolymerization factors (Lappalainen & Drubin, 1997, Rodal et al., 1999, Chen et al., 2015, Michelot & Drubin, 2011, Okreglak & Drubin, 2010). Due to the ability in binding to the branched actin filament, we can use the fluorescent signal of Abp1-RFP imaged by fluorescent wide-field microscopy to trace the assembly and disassembly of actin filaments during endocytosis from each cortical patch (Fig. 2C). On average, the lifetime of actin patches labelled by Abp1-RFP is 10s. In such short window of time, assembled actin network generates sufficient mechanical force to facilitate the internalization of endocytic pits from the plasma membrane. Upon a heat stress treatment, a general increase of endocytosis efficiency will be observed, in which the lifetime of actin patches labelled by Abp1-RFP decreases to 8s on average (Fig. 2D). Heat stress also affected the polarized localization of endocytic sites in the cell. Under room temperature, the cortical patches are largely concentrated in the bud, but when the temperature increases to 39°C within 30 minutes, the actin patches were depolarized and showed more endocytic sites in the mother cell around the cell periphery (Fig. 2A). Actin patches will relocate back to the bud after 30 min despite the persistence of the heat treatment, which is a depolarization and repolarization phenomena for polarity proteins and actin cytoskeleton that are involved in multiple signaling pathways (Levin, 2011, Guo et al., 2009). To compare the actin cytoskeleton at the polarized and depolarized stages, we imaged the cells at 20 min heat stress condition. Under heat stress, actin patches are depolarized, and actin cables are less defined and undergo an architectural reorganization, suggesting the changes in coordinated actin filament assembly. The Arabidopsis root hair serves as an ideal model to study actin cytoskeleton regulation in a polarized plant cell. The fast polarized cell growth highly depends on the polarized organization of cytoplasmic actin networks (Carol & Dolan, 2002). Dynamic actin filaments assembly and disassembly are critical for supporting fast polarized cell growth of plant root hair. Actin filament bundles create stable networks for vesicular trafficking and generating mechanical forces for polarized cell growth. Interestingly, actin filament in different positions of polarized root hair cells shows different dynamic behaviors. Those actin filaments in the base region are relatively more stable with a higher level of actin bundles, revealed by the quantitative measurement of actin filament diameter and mean intensity (Fig. 3C and 3D). Due to the limitation in resolution of widefield microscopy, actin filament diameter is less informative in general to indicate the precise actin bundle structure. Therefore, an increase of filament diameter from the tip to base root hair region does not represent a linear increase of actin filament number in each bundle, though a relative modulation of bundling level is effectively presented. Such difference between different zones of root hair could be explained by the cellular content at each region. In the root hair base region, most of the cytoplasm is occupied by a central vacuole, and no cell expansion happens in the region (Ketelaar, 2013). Plant protein sorting and vesicular delivery are involved in multiple intracellular activities of membrane transport and cell growth (Miao et al., 2011, Miao & Jiang, 2007, Miao et al., 2008, Miao et al., 2006, Wang et al., 2009, Kim et al., 2010, Min et al., 2007). The highly bundled actin cytoskeleton serves as a back bone to maintain the cell shape and is involved in the long-distance delivering of vesicles to the root hair tip. However, with the extension to tips, the actin filaments disperse to thinner and more dynamic filaments gradually. In the fast-growing tip, the thinner and dynamic actin filaments allow the fast exchange of membranes between growing tip and intracellular organelles (Pei et al., 2012). Such different actin dynamics at different cell positions may also suggest different stabilization in actin network formation, which worth of future studies in understanding the mechanisms by which the actin network assembly are well-cooperated
by actin-binding proteins during polarized root hair growth. However, using Lifeact as actin filament marker require careful design and caution in results interpretation. mCherry fluorescent protein fused Liftact was indicated to have potential perturbation in actin filament polymerization in a concentration-dependent manner (Courtemanche et al., 2016). An appropriate selection of fluorescent probe and the control of fluorescent protein expression level should be taken into consideration in experimental design. 7.2
Technique considerations for imaging and image analysis 7.2.1
Improving image resolution and contrast during acquisition
Most budding yeast strains are auxotrophic and the growth media needs to be supplemented with essential amino acids. However, some amino acid can hinder and increase the background fluorescence such as tryptophan. Hence it is advised to do imaging using growth media without tryptophan. In addition, to achieve high image resolution and quality, we use immersion oil with a refractive index of n= 1.518 to match the refractive index of the imaging medium and cover glass (thickness, 0.17 mm). 7.2.2
Consideration of using deconvolution to improve image quality
Raw images collected by the epifluorescence microscope can give high background noise caused by blurring from diffraction and photon noise, especially when imaging samples with dim signals. Applying deconvolution can help to enhance the signal to noise ratio by removing unwanted background while improving the signal of interest. This works the best for analyzing lifetime of actin patches as it improves the contrast of kymographs of the lifetime of yeast endocytic proteins before data analysis. However, not all images are suitable for deconvolution. Original images with high background noise or weak fluorescence signal are not advised for deconvolution. Only apply deconvolution to improve the resolution and contrast while not compromising the integrity of images. Below are some tips to consider before deconvolution: I. Remove unwanted slices in a z-stacks that are often out of plane or focus on improving the deconvolution results; II. For budding yeast analysis, crop out regions of interest that has better signal and disregard areas with larger background noise can improve the final resolution. 8. Acknowledgements This work was supported by grants from Nanyang Technological University NAP start-up grant, NIMBELS (NIM/01/2016), and MOE Tier 2 (MOE2016-T2-1-005) to Y. MIAO. 9. References Cao L, Henty‐Ridilla JL, Blanchoin L, Staiger CJ, 2016. Profilin‐Dependent Nucleation and Assembly of Actin Filaments Controls Cell Elongation in Arabidopsis. Plant Physiol 170, 220‐33. Carol RJ, Dolan L, 2002. Building a hair: tip growth in Arabidopsis thaliana root hairs. Philosophical Transactions of the Royal Society of London B: Biological Sciences 357, 815‐21.
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