The authors are grateful to Julie Merkle for her helpful comments on the manuscript. This work was supported by NIH grant R15GM137236 to O.D. The confocal and super-resolution images were acquired using a Zeiss LSM 900 with Airyscan 2, purchased with NSF MRI grant 2018748.

The authors have nothing to disclose.

The BM is critical for embryonic and organ morphogenesis, and adult physiological functions. Moreover, the BM acts as a signaling platform for the establishment and maintenance of epithelial polarity and provides tissues with support2. Yet, the mechanisms that regulate the proper placement of BM proteins are poorly understood. A better understanding of the biological pathways dedicated to the intracellular trafficking and polarized secretion of BM proteins requires a careful analysis of the compon...

The basement membrane (BM) is a thin sheet of layered cell-adherent extracellular matrix (ECM) critical for epithelial structure and morphogenesis1. It comprises ~50 proteins and is found ubiquitously underlying the epithelial and endothelial cells, and ensheathing skeletal, smooth, and heart muscle cells and adipocytes1,2,3. The three main components of the BM at the basal side of the epithelial cells are Collagen IV, Perlecan, and Laminins. The BM underlies the epithelial cells and is responsible for many functions, including tissue separation and barrier, growth and support, and cell polarization2,3,4,5,6,7,8,9,10,11,12. Its role as a signaling platform regulates the morphology and differentiation of epithelial cells and tissues during development3,13,14. Moreover, the mis-regulation of the BM and/or a breach in its integrity are the primary causes of many pathological conditions, including tumor metastasis2,15,16. Despite the essential functions performed by the BM during tissue and organ morphogenesis, the components of the biological pathway(s) dedicated to the polarized intracellular trafficking and secretion of BM proteins are vaguely known.
To study the intracellular trafficking of BM-containing vesicles and the secretion of BM proteins by epithelial cells, the follicular epithelium (FE) of the Drosophila ovary is a powerful model system (Figure 1). A Drosophila ovary comprises 16-20 long, tube-like structures, called ovarioles (Figure 1A,B)17,18,19. Each ovariole can be thought of as an egg assembly line, with the age progression of egg chambers (which each gives rise to an egg) that begins at the anterior end and moves posteriorly, until the mature egg exits through the oviduct. Each egg chamber is encapsulated by the FE, a monolayer of somatic follicle cells (FCs), that surrounds the central germline cells (GCs). The FE is highly polarized with a distinct apical-basal polarity where the apical domain faces the germline, and the BM proteins are secreted basally18,19. The FCs actively secrete all of the major components of the BM, including Collagen IV, Perlecan, and Laminins20,21. In epithelial cells such as FCs, the BM components are produced and require a specialized polarized secretion pathway for their deposition extracellularly. For example, in the case of the most abundant component of the BM, Collagen IV (Coll IV), the details surrounding its polarized intracellular trafficking and secretion are vague despite its production and deposition being the focus of many studies. Coll IV is translated in the endoplasmic reticulum (ER), which is also where each fibril - composed of three polypeptides (two &#945;1 chains and one &#945;2 chain) - is assembled into a triple helix22. Proper Coll IV folding and function require ER chaperones and enzymes, including lysyl and prolyl-hydroxylases such as Plod and PH4&#945;EFB20,22,23,24,25,26. These posttranslational enzymes regulate the ER sorting of Coll IV, as the loss of each causes Coll IV to be trapped in the basal ER20,23,24,25,26. Then, newly synthesized Coll IV exits the ER for the Golgi in COPII-coated vesicles. The cargo receptor Tango1 aids in packaging collagens into sizable Golgi-bound vesicles that can accommodate large multimeric proteins20,27. Once Coll IV is packaged into intracellular exocytic vesicles, it is specifically secreted basally from epithelial cells. To direct BM deposition to the basal side, epithelial cells require another set of factors specifically dedicated to polarized BM secretion. Using the FE of the Drosophila ovary, a few components of this novel cellular process have been characterized, including the nucleotide exchange factors (GEFs) Crag and Stratum, the GTPases Rab8 and Rab10, as well as the levels of the phosphoinositide PI(4,5)P2, and Kinesin 1 and 3 motor proteins20,28,29,30,31. These components are critical in ensuring the polarized distribution of BM proteins.
To monitor the intracellular localization of BM proteins in the FE, endogenously tagged basement membrane proteins (protein traps), such as Viking-GFP (Vkg-GFP or &#945;2 Coll IV-GFP) and Perlecan-GFP (Pcan-GFP) can be used32,33. These protein trap lines have been shown to accurately reflect the endogenous distribution of BM proteins and allow for more sensitive detection of vesicular trafficking28,30. The components involved in the polarized deposition of BM in the FE were first characterized using protein trap lines for Vkg-GFP and Pcan-GFP20,28,29,30. Protein traps can be used in different genetic backgrounds, including mutants and Gal4 lines34. Moreover, protein traps can be used in combination with fluorescent dyes and/or fluorescence immunostaining, allowing for precise characterization of the localization of BM proteins when comparing wild-type and mutant conditions35.
To accurately and efficiently assess the distribution and localization of BM protein-containing vesicles, confocal laser scanning microscopy (CLSM) and super-resolution imaging techniques present a significant advantage to other imaging approaches. These approaches couple high-resolution imaging with relative ease of use. CLSM is a microscopy technique that allows for an improved optical resolution by scanning the specimen with a laser in a raster scan manner using galvanometers. The pinhole aperture is a core component of a confocal microscope. By blocking the out-of-focus signals coming from above or below the focal plane, the pinhole aperture results in a highly superior resolution in the z-axis36. This also makes it possible to obtain a series of images in the z-plane, called a z-stack, corresponding to a series of optical sections. z-stacks subsequently create a 3D image of the specimen, via 3D reconstruction, with the aid of imaging software. Conventional epifluorescence (widefield) microscopes, unlike confocal microscopes, allow out-of-focus light to contribute to image quality, decreasing image resolution and contrast36,37. This makes epifluorescence microscopy a less attractive candidate when studying protein localization or colocalization.
Although CLSM is a suitable approach for various applications, including imaging and characterization of the intracellular trafficking of BM proteins, it still presents an issue when imaging samples below Abbe&#39;s diffraction limit of light (200-250 nm). When imaging such samples, confocal microscopy, especially when using an oil objective, can result in high resolution. However, super-resolution techniques surpass the limit of confocal microscopy. There are various approaches to achieve super-resolution microscopy, each with specific resolution limits, and each appropriate for different analyses. These approaches include photoactivated localization microscopy (PALM) or stochastic optical reconstruction microscopy (STORM), stimulated emission depletion microscopy (STED), structured illumination microscopy (SIM), and Airyscan (super-resolution) microscopy38,39,40,41,42,43,44,45,46. Although Airyscan has a coarser resolution than PALM/STORM, STED, and SIM, it can still achieve a resolution of up to ~120 nm (about twice the resolution of CLSM). Furthermore, this super-resolution microscopy approach has been shown to have an advantage over SIM and other super-resolution techniques when imaging thick samples and samples with a low signal-to-noise ratio47,48.
Airyscan is a relatively new super-resolution confocal microscopy technology46. Unlike traditional CLSMs, which use the pinhole and single point detectors to reject out-of-focus light, this super-resolution approach uses a 32-channel gallium arsenide phosphide (GaAsP) photomultiplier tube area detector that collects all of the light at every scan position45. Each of the 32 detectors work as a small pinhole, reducing the pinhole size from the traditional 1.0 Airy Unit (A.U.) to an enhanced 0.2 A.U., enabling an even higher resolution and signal-to-noise ratio, while maintaining the efficiency of a 1.25 A.U. diameter45. Furthermore, the linear deconvolution used by Airyscan results in up to a 2x increase in resolution45. Taking this into consideration, CLSM, and specifically super-resolution microscopy, are&#160;well-suited to study BM proteins and proteins that regulate the basal deposition of BM proteins, as they can produce very high-resolution images for localization and colocalization studies, thereby providing new insights in the spatial, temporal, and molecular events that control these processes.
An alternative approach to confocal microscopy that can be used to perform localization experiments is image deconvolution. Since widefield microscopy allows out-of-focus light to reach the detectors, mathematical and computational deconvolution algorithms can be applied to remove or reassign out-of-focus light from images obtained by widefield microscopy, thereby improving the resolution and contrast of the image49. Deconvolution algorithms can also be applied to confocal images to further increase resolution and contrast, producing final images almost comparable to that of super-resolution microscopy50. Airyscan makes use of Weiner filter-based deconvolution along with Sheppard&#39;s pixel reassignment, resulting in a highly improved spatial resolution and signal-to-noise ratio. Compared to confocal microscopy, an increase of 2x in resolution in all three spatial dimensions (120 nm in x and y, and 350 nm in z) is observed when using this super-resolution microscopy technique45,51.
This manuscript provides detailed and optimized protocols to stain, acquire, and visualize the intracellular trafficking and deposition of BM proteins using the FE of the Drosophila ovary as a model system coupled with confocal and super-resolution microscopy. Drosophila lines expressing endogenously tagged basement membrane proteins, e.g., Vkg-GFP and Pcan-GFP, are efficient and accurate tools to visualize BM protein trafficking and secretion. In addition, they can be easily used in different genetic backgrounds, including mutant and Gal4/UAS lines34. Although endogenously tagged basement membrane proteins are recommended, the use of antibodies against specific BM proteins is also compatible with the described protocols. These protocols are particularly useful for scientists who are interested in studying intracellular trafficking and the secretion of BM proteins in intact epithelial tissue using confocal and super-resolution imaging. Moreover, the ability to combine epithelial tissue analysis with the expansive tools of Drosophila genetics makes this approach especially powerful. Finally, these protocols could be easily adapted to study vesicular trafficking and sorting of other proteins of interest.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa Fluor 546 phalloidin</td>
			<td>Invitrogen</td>
			<td>A22283</td>
			<td>F-Actin Stain (1/500 of 66&#181;M)</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 phalloidin</td>
			<td>Invitrogen</td>
			<td>A22287</td>
			<td>F-Actin Stain (1/100 of 66&#181;M))</td>
		</tr>
		<tr>
			<td>Anti-GM130 Antibody</td>
			<td>abcam</td>
			<td>ab30637</td>
			<td>For Golgi Stain (colocalization); use as concentration of 7&#181;g/uL</td>
		</tr>
		<tr>
			<td>Aqua-Polymount</td>
			<td>Polysciences, Inc.</td>
			<td>1860620</td>
			<td>Mounting Medium</td>
		</tr>
		<tr>
			<td>Bakers Yeast (Active Dry Yeast)</td>
			<td>Genesee Scientific</td>
			<td>62-103</td>
			<td>To fatten the overies for dissection</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (30% solution)</td>
			<td>Sigma-Aldrich</td>
			<td>A7284</td>
			<td>For blocking solution</td>
		</tr>
		<tr>
			<td>Depression wells</td>
			<td>Electron Microscopy Sciences</td>
			<td>7156101</td>
			<td>For dissection (glass concavity slide can be used instead)</td>
		</tr>
		<tr>
			<td>Dissecting needle</td>
			<td>Fisher scientifc</td>
			<td>13-820-024</td>
			<td></td>
		</tr>
		<tr>
			<td>Drosophila Incubator</td>
			<td>Genesee Scientific/Invictus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fly Stock: Perlecan-GFP Drosophila line (ZCL1700)</td>
			<td></td>
			<td></td>
			<td>Morin et al., 2001</td>
		</tr>
		<tr>
			<td>Fly Stock: UAS-Crag RNAi line (TRIP line HMS00241)</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>33594</td>
			<td>RNAi against Crag</td>
		</tr>
		<tr>
			<td>Fly Stock: Viking-GFP Drosophila line (CC00791)</td>
			<td></td>
			<td></td>
			<td>Buszczak et al., 2007</td>
		</tr>
		<tr>
			<td>Fly Stock: Vkg-GFP, tj-Gal4</td>
			<td></td>
			<td></td>
			<td>Devergne et al., 2017. Drive the expression of Crag RNAi in the FE</td>
		</tr>
		<tr>
			<td>Forceps (Dumont 5)</td>
			<td>Fine Science Tools</td>
			<td>11251-30</td>
			<td>For dissection</td>
		</tr>
		<tr>
			<td>Glass Concavity Slide</td>
			<td>Electron Microscopy Sciences</td>
			<td>7187804</td>
			<td>For dissection (depression wells can be used instead)</td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG, Alexa Fluor 568</td>
			<td>Invitrogen</td>
			<td>A11036</td>
			<td>Secondary antibody (GM130 antibody) (5 &#181;g/mL)</td>
		</tr>
		<tr>
			<td>Hoechst (Hoechst 33342)</td>
			<td>Invitrogen</td>
			<td>H3570</td>
			<td>DNA Stain (1 ug/mL)</td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimtech</td>
			<td>Fisher Scientific: 06-666</td>
			<td>Delicate task wipers</td>
		</tr>
		<tr>
			<td>Leica Fluorescent Stereo Microscope&#160; M165 FC</td>
			<td>Leica</td>
			<td></td>
			<td>For ovary imaging</td>
		</tr>
		<tr>
			<td>Microscope Slides</td>
			<td>Corning</td>
			<td>294875X25</td>
			<td>Microscope Slides</td>
		</tr>
		<tr>
			<td>Nutating platform rocker</td>
			<td>Corning Life Sciences</td>
			<td>6720</td>
			<td>For ovary fixation and staining</td>
		</tr>
		<tr>
			<td>Nutri-Fly BF</td>
			<td>Genesee Scientific</td>
			<td>66-121</td>
			<td>Fly Food</td>
		</tr>
		<tr>
			<td>Paraformaldehyde 20% Solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>Fisher Scientific: 15713</td>
			<td>For PFA 4%</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline Tablets</td>
			<td>Fisher scientific</td>
			<td>BP2944100</td>
			<td>For PBS solution</td>
		</tr>
		<tr>
			<td>ProLong Glass Antifade Mountant</td>
			<td>Invitrogen</td>
			<td>P36980</td>
			<td>Mounting Medium</td>
		</tr>
		<tr>
			<td>Square Cover Glass</td>
			<td>Corning</td>
			<td>285022</td>
			<td>Cover glass for microscope slides</td>
		</tr>
		<tr>
			<td>Triton x-100</td>
			<td>Sigma-Aldrich</td>
			<td>9036-19-5</td>
			<td>For PBST</td>
		</tr>
		<tr>
			<td>Zeiss LSM 900 with Airyscan 2</td>
			<td>Zeiss</td>
			<td></td>
			<td>Confocal and super-resolution Microscope</td>
		</tr>
		<tr>
			<td>Zeiss Stemi 305 Stereo Microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>Dissecting microscope</td>
		</tr>
		<tr>
			<td>Zeiss Zen Software version 3.3 (Blue Edition)</td>
			<td>Zeiss</td>
			<td></td>
			<td>Image acquisition and processing</td>
		</tr>
	</tbody>
</table>

confocal and super-resolution imaging of polarized intracellular trafficking and secretion of basement membrane proteins during drosophila oogenesis

The basement membrane (BM) - a specialized sheet of extracellular matrix present at the basal side of epithelial cells - is critical for the establishment and maintenance of epithelial tissue morphology and organ morphogenesis. Moreover, the BM is essential for tissue modeling, serving as a signaling platform, and providing external forces to shape tissues and organs. Despite the many important roles that the BM plays during normal development and pathological conditions, the biological pathways controlling the intracellular trafficking of BM-containing vesicles and how basal secretion leads to the polarized deposition of BM proteins are poorly understood. The follicular epithelium of the Drosophila ovary is an excellent model system to study the basal deposition of BM membrane proteins, as it produces and secretes all major components of the BM. Confocal and super-resolution imaging combined with image processing in fixed tissues allows for the identification and characterization of cellular factors specifically involved in the intracellular trafficking and deposition of BM proteins. This article presents a detailed protocol for staining and imaging BM-containing vesicles and deposited BM using endogenously tagged proteins in the follicular epithelium of the Drosophila ovary. This protocol can be applied to address both qualitative and quantitative questions and it was developed to accommodate high-throughput screening, allowing for the rapid and efficient identification of factors involved in the polarized intracellular trafficking and secretion of vesicles during epithelial tissue development.

The methods described herein can be used to efficiently and accurately image and characterize the intracellular trafficking and secretion of BM proteins in polarized epithelial cells, such as the FE of the Drosophila ovary. Next, we provide anticipated results obtained using the described methods, as well as helpful advice and potential pitfalls. To do so, Vkg-GFP, an endogenously tagged Vkg (Drosophila Col IV) is used. However, the same results can be achieved with other endogenously tagged BM proteins...

Watch this Scientific Journal Video about Confocal and Super-Resolution Imaging of Polarized Intracellular Trafficking and Secretion of Basement Membrane Proteins During Drosophila Oogenesis at JoVE.c

Confocal and Super-Resolution Imaging of Polarized Intracellular Trafficking and Secretion of Basement Membrane Proteins During Drosophila Oogenesis

The basement membrane is essential for tissue and organ morphogenesis during development. To better understand the mechanisms leading to proper placement of this structure, the protocol presented describes methods to visualize and characterize the intracellular trafficking and secretion of basement membrane proteins in epithelial cells using confocal and super-resolution microscopy.

Confocal and Super-Resolution Imaging of Polarized Intracellular Trafficking and Secretion of Basement Membrane Proteins During Drosophila Oogenesis

The basement membrane (BM) - a specialized sheet of extracellular matrix present at the basal side of epithelial cells - is critical for the establishment ...

confocal-super-resolution-imaging-polarized-intracellular-trafficking

Research

JoVE Journal

Developmental Biology

2.1K Views.  Northern Illinois University. The basement membrane is essential for tissue and organ morphogenesis during development. To better understand the mechanisms leading to proper placement of this structure, the protocol presented describes methods to visualize and characterize the intracellular trafficking and secretion of basement membrane proteins in epithelial cells using confocal and super-resolution microscopy.

Video: Confocal and Super-Resolution Imaging of Polarized Intracellular Trafficking and Secretion of Basement Membrane Proteins During Drosophila Oogenesis

Live Confocal Imaging of Developing Arabidopsis Flowers

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. Recent advances in confocal microscopy, combined with the development of numerous fluorophores, have overcome these issues and opened the possibility to study the expression of several genes simultaneously, with a good cellular resolution, in live samples. Live confocal imaging provides plant biologists with a powerful tool to study development, and has been extensively used to study root growth and the formation of lateral organs on the flanks of the shoot apical meristem. However, it has not been widely applied to the study of flower development, in part due to challenges that are specific to imaging flowers, such as the sepals that grow over the flower meristem, and filter out the fluorescence from underlying tissues. Here, we present a detailed protocol to perform live confocal imaging on live, developing Arabidopsis flower buds, using either an upright or an inverted microscope.

Live confocal imaging provides biologists with a powerful tool to study development. Here, we present a detailed protocol for the live confocal imaging of developing Arabidopsis flowers.

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. ...

A Simple Chamber for Long-term Confocal Imaging of Root and Hypocotyl Development

Several aspects of plant development, such as lateral root morphogenesis, occur on time spans of several days. To study underlying cellular and subcellular processes, high resolution time-lapse microscopy strategies that preserve physiological conditions are required. Plant tissues must have adequate nutrient and water supply with sustained gaseous exchange but, when submerged and immobilized under a coverslip, they are particularly susceptible to anoxia. One strategy that has been successfully employed is the use of a perfusion system to maintain a constant supply of oxygen and nutrients. However, such arrangements can be complicated, cumbersome, and require specialized equipment. Presented here is an alternative strategy for a simple imaging system using perfluorodecalin as an immersion medium. This system is easy to set up, requires minimal equipment, and is easily mounted on a microscope stage, allowing several imaging chambers to be set up and imaged in parallel. In this system, lateral root growth rates are indistinguishable from growth rates under standard conditions on agar plates for the first two days, and lateral root growth continues at reduced rates for at least another day. Plant tissues are supplied with nutrients via an agar slab that can be used also to administer a range of pharmacological compounds. The system was established to monitor lateral root development but is readily adaptable to image other plant organs such as hypocotyls and primary roots.

Presented here is a simple technique for high-resolution confocal time-lapse imaging of root and hypocotyl development for up to 3 days using high numerical-aperture objectives and perfluorodecalin as an immersion medium.

Several aspects of plant development, such as lateral root morphogenesis, occur on time spans of several days. To study underlying cellular and ...

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended intracellular endotubes that build and stabilize the lumen with a membrane and submembraneous cytoskeleton of apical character. The excretory cell expands its length approximately 2,000 times to generate these canals, making this model unique for the in vivo assessment of de novo polarized membrane biogenesis, intracellular lumen morphogenesis and unicellular tubulogenesis. The protocol presented here shows how to combine standard labeling, gain- and loss-of-function genetic or RNA interference (RNAi)-, and microscopic approaches to use this model to visually dissect and functionally analyze these processes on a molecular level. As an example of a labeling approach, the protocol outlines the generation of transgenic animals with fluorescent fusion proteins for live analysis of tubulogenesis. As an example of a genetic approach, it highlights key points of a visual RNAi-based interaction screen designed to modify a gain-of-function cystic canal phenotype. The specific methods described are how to: label and visualize the canals by expressing fluorescent proteins; construct a targeted RNAi library and strategize RNAi screening for the molecular analysis of canal morphogenesis; visually assess modifications of canal phenotypes; score them by dissecting fluorescence microscopy; characterize subcellular canal components at higher resolution by confocal microscopy; and quantify visual parameters. The approach is useful for the investigator who is interested in taking advantage of the C. elegans excretory canal for identifying and characterizing genes involved in the phylogenetically conserved processes of intracellular lumen and unicellular tube morphogenesis.

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging

The C. elegans excretory canal is a unique single-cell model for the visual in vivo analysis of de novo polarized membrane biogenesis. This protocol describes a combination of standard genetic/RNAi and imaging approaches, adaptable for the identification and characterization of molecules directing unicellular tubulogenesis, and apical membrane and lumen biogenesis.

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended ...

Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and scanning electron microscopy. The sub-diffraction limit resolution capabilities of super-resolution light microscopes allow improving the accuracy of the correlated data. Briefly, 110 nanometer thick cryo-sections are transferred to a silicon wafer and, after immunofluorescence staining, are imaged by super-resolution light microscopy. Subsequently, the sections are preserved in methylcellulose and platinum shadowed prior to imaging in a scanning electron microscope (SEM). The images from these two microscopy modalities are easily merged using tissue landmarks with open source software. Here we describe the adapted method for the larval zebrafish retina. However, this method is also applicable to other types of tissues and organisms. We demonstrate that the complementary information obtained by this correlation is able to resolve the expression of mitochondrial proteins in relation with the membranes and cristae of mitochondria as well as to other compartments of the cell.

This protocol describes the necessary steps to obtain subcellular protein localization results on zebrafish retina by correlating super-resolution light microscopy and scanning electron microscopy images.

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and ...

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and differentiation. Typically, pHi dynamics are determined in cultured cells, which are amenable to measuring and experimentally manipulating pHi. However, the recent development of new tools and methodologies has made it possible to study pHi dynamics within intact, live tissue. For Drosophila research, one important development was the generation of a transgenic line carrying a pHi biosensor, mCherry::pHluorin. Here, we describe a protocol that we routinely use for imaging live Drosophila ovarioles to measure pHi in the epithelial follicle stem cell (FSC) lineage in mCherry::pHluorin transgenic wild type lines; however, the methods described here can be easily adapted for other tissues, including the wing discs and eye epithelium. We describe techniques for expressing mCherry::pHluorin in the FSC lineage, maintaining ovarian tissue during live imaging, and acquiring and analyzing images to obtain pHi values.

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

We provide a protocol for imaging intracellular pH of an epithelial stem cell lineage in live Drosophila ovarian tissue. We describe methods to generate transgenic flies expressing a pH biosensor, mCherry::pHluorin, image the biosensor using quantitative fluorescence imaging, generate standard curves, and convert fluorescence intensity values to pH values.

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and ...

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

Analysis of Epididymal Protein Synthesis and Secretion

The mammalian epididymis generates one of the most complex intraluminal fluids of any endocrine gland in order to support the post-testicular maturation and storage of spermatozoa. Such complexity arises due to the combined secretory and absorptive activity of the lining epithelial cells. Here, we describe the techniques for the analysis of epididymal protein synthesis and secretion by focusing on the model protein family of dynamin (DNM) mechanoenzymes; large GTPases that have the potential to regulate bi-directional membrane trafficking events. For the study of protein expression in epididymal tissue, we describe robust methodology for immunofluorescence labeling of target proteins in paraffin-embedded sections and the subsequent detection of the spatial distribution of these proteins via immunofluorescence microscopy. We also describe optimized methodology for the isolation and characterization of exosome like vesicles, known as epididymosomes, which are secreted into the epididymal lumen to participate in intercellular communication with maturing sperm cells. As a complementary approach, we also describe the immunofluorescence detection of target proteins in an SV40-immortalized mouse caput epididymal epithelial (mECap18) cell line. Moreover, we discuss the utility of the mECap18 cell line as a suitable in vitro model with which to explore the regulation of epididymal secretory activity. For this purpose, we describe the culturing requirements for the maintenance of the mECap18 cell line and the use of selective pharmacological inhibition regimens that are capable of influencing their secretory protein profile. The latter are readily assessed via harvesting of conditioned culture medium, concentration of secreted proteins via trichloroacetic acid/acetone precipitation and their subsequent analysis via SDS-PAGE and immunoblotting. We contend that these combined methods are suitable for the analysis of alternative epididymal protein targets as a prelude to determining their functional role in sperm maturation and/or storage.

Here, we report the immunofluorescence localization of dynamin to illustrate the protocols for the detection of proteins in paraffin-embedded mouse epididymal sections and those of an immortalized epididymal cell line (mECap18). We also describe the protocols for the isolation of secretory proteins from both epididymal fluid and conditioned cell media.

The mammalian epididymis generates one of the most complex intraluminal fluids of any endocrine gland in order to support the post-testicular maturation ...

Confocal Live Imaging of Shoot Apical Meristems from Different Plant Species

The shoot apical meristem (SAM) functions as a conserved stem cell reservoir and it generates almost all aboveground tissues during the postembryonic development. The activity and morphology of SAMs determine important agronomic traits, such as shoot architecture, size and number of reproductive organs, and most importantly, grain yield. Here, we provide a detailed protocol for analyzing both the surface morphology and the internal cellular structure of the living SAMs from different species through laser scanning confocal microscope. The whole procedure from the sample preparation to the acquisition of high resolution three-dimensional (3D) images can be accomplished within as short as 20 minutes. We demonstrate that this protocol is highly efficient for studying not only the inflorescence SAMs of the model species but also the vegetative meristems from different crops, providing a simple but powerful tool to study the organization and development of meristems across different plant species.

This protocol presents how to live image and analyze the shoot apical meristems from different plant species using laser scanning confocal microscopy.

The shoot apical meristem (SAM) functions as a conserved stem cell reservoir and it generates almost all aboveground tissues during the postembryonic ...

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits are required to control this behavior. Failure to produce the rhythmic pattern of contractions can be indicative of neurological abnormalities. We previously found that defects in protein O-mannosylation, a posttranslational protein modification, affect the axon morphology of sensory neurons and result in abnormal coordinated muscle contractions in embryos. Here, we present a relatively simple method for recording and analyzing the pattern of peristaltic muscle contractions by live imaging of late stage embryos up to the point of hatching, which we used to characterize the muscle contraction phenotype of protein O-mannosyltransferase mutants. Data obtained from these recordings can be used to analyze muscle contraction waves, including frequency, direction of propagation and relative amplitude of muscle contractions at different body segments. We have also examined body posture and taken advantage of a fluorescent marker expressed specifically in muscles to accurately determine the position of the embryo midline. A similar approach can also be utilized to study various other behaviors during development, such as embryo rolling and hatching.

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Here, we present a method to record embryonic muscle contractions in Drosophila embryos in a non-invasive and detail-oriented manner.

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits ...

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the number of novel cell lines is expected to increase. The DGRC aims to familiarize researchers with using Drosophila cell lines as an experimental tool to complement and drive their research agenda. Procedures for working with a variety of Drosophila cell lines with distinct characteristics are provided, including protocols for thawing, culturing, and cryopreserving cell lines. Importantly, this publication demonstrates the best practices required to work with Drosophila cell lines to minimize the risk of contaminations from adventitious microorganisms or from other cell lines. Researchers who become familiar with these procedures will be able to delve into the many applications that use Drosophila cultured cells including biochemistry, cell biology and functional genomics.

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Drosophila cell lines are important reagents for both fundamental and biomedical research. This article provides protocols for thawing, subculturing, and the cryopreservation of commonly used Drosophila cell lines to assist researchers in incorporating the use of these reagents in their research.

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the ...