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 components of these pathways and their roles in BM processing. One way to achieve this is to use confocal and super-resolution imaging. Here, we described methods for the preparation and staining or immunostaining of Drosophila ovaries to efficiently image the intracellular trafficking and secretion of BM proteins in the FE using confocal and super-resolution microscopy.
Advantages of protein trap and endogenously tagged proteins to visualize the intracellular trafficking and secretion of BM proteins in wild-type and mutant conditions 
The provided protocol takes full advantage of protein traps and endogenously tagged BM proteins (e.g., Vkg-GFP and Pcan-GFP) to image and assess the localization and distribution of BM proteins in the FE in wild-type (control) and mutant conditions. These lines have important advantages over the use of antibodies against BM proteins. First, antibodies against specific proteins of interest are often rare and in low abundance. In addition, there are often issues with tissue penetrance associated with antibodies which can lead to inaccurate observation and assessment of the protein of interest. Moreover, protein trap lines can be inserted into different genetics background used to determine the functions of factors required for the proper deposition of the BM such as (i) methods to manipulate gene expression (e.g., UAS/Gal4), (ii) mutants, and (iii) RNA interference (RNAi) lines. Finally, these protein traps can be used to visualize the localization and function of BM using live imaging57.
However, the fusion of a fluorescent tag to a protein of interest can interfere with its localization and functions. Thus, it is critical to evaluate any aberrant effects of the tag on the protein. One way to ensure that the addition of a fluorescent tag does not interfere with the function and localization of BM proteins is to determine whether the transgenic Drosophila line is homozygous viable. Both the Vkg-GFP and Pcan-GFP lines that are used in the different experiments described here and previously, are homozygous viable, indicating that the addition of a GFP tag does not interfere with the function of these essential proteins29,30.
Importance of tissue preparation, fixation and staining for imaging 
To efficiently and accurately image the FE, it is critical to prepare, fix, and stain the ovarian tissue properly as directed in this method. First, after dissection, carefully remove all other organs and fly debris before fixation. Using paraformaldehyde (PFA) to fix tissue is recommended, since it leads to bright GFP fluorescence and is compatible with most antibodies. However, PFA may not be compatible with all antibodies; some may require glutaraldehyde or methanol fixation. Moreover, to avoid background fluorescence due to non-specific binding of the primary antibody, the ovarian tissue should be incubated on a nutating platform rocker for at least 1 h in blocking solution, and&#160;extensively washed several times after primary and secondary antibody incubation as described in the protocol. Finally, proper mounting of the ovarian tissue is essential to achieve optimal imaging. This requires careful separation of individual egg chambers to avoid their stacking on top of each other. It is also important to let the mounting medium fully polymerize before imaging to avoid floating of the tissue under the coverslip. This further allows the mounting media to reach its optimal reflective index, which is very important for super-resolution imaging. Failing to do so will affect the image quality. Furthermore, the prepared slides can be stored for up to 1 month at 4 &#176;C, before the fluorescence is quenched. In addition to the provided protocol, several other protocols are available for ovarian dissection and staining53,54,55.
The use of proper acquisition and processing parameters is critical for optimal imaging and to assess the localization of BM proteins using confocal and super-resolution imaging 
As described previously, it is critical to properly set the acquisition parameters for confocal and super-resolution imaging to obtain optimal images of the specimen. For image acquisition, the ROI must be carefully selected using the zoom function, and then setting the frame size and acquisition speed optimally. Moreover, as illustrated in Figure 2, it is critical to adjust the laser power and the detector gain appropriately to visualize intracellular trafficking without saturating the detectors. These parameters must be set very carefully to avoid underexposed or overexposed images (Figure 2). Underexposed images will result in low resolution images, in which the intracellular structures will be difficult to visualize and characterize (Figure 2B). Overexposed images due to the saturation of the detector lead to data misinterpretation, and colocalization artifacts (Figure 2C). If the aim is to determine the vesicular localization of endogenously tagged BM proteins, the fluorescence from GFP-tagged proteins (e.g., Vkg-GFP and Pcan-GFP) in the basal BM quickly saturates the detector. However, vesicles containing less GFP-tagged BM proteins will appear dim. Hence, it is important to set the image sensitivity using the BM-containing intracellular structures (e.g., vesicles), and not on the BM (Figure 2). Selecting pinhole size is also very important. Ideally, a pinhole size of 1 AU for each channel should be selected for best quality images, especially when the resolution in the z-axis is important. The smaller pinhole size is optimal for thinner optical sections. However, when optimal z-axis resolution is not required or a z-stack is not being captured, the pinhole size can be increased to avoid photobleaching. Furthermore, for very dim signals, increasing the pinhole size may help due to a higher signal-to-noise ratio and the capture of more photons, aiding in data interpretation.
For super-resolution imaging, particular attention should be taken for deconvolution processing to avoid generating under- or over-processed images that are poorly resolved, as illustrated in Figure 3. Finally, to achieve an optimal 3D reconstruction, setting the best interval determined by the software is strongly recommended. An interval too large between z-sections (i.e., higher than 0.5 &#181;m) will lead to poor 3D rendering and will affect the subsequent analysis of the phenotype.
Advantage of super-resolution over traditional confocal microscopy when imaging BM intracellular trafficking 
As illustrated in the results section, although the localization and distribution of BM proteins can be accurately assessed using confocal imaging, the super-resolution approach described generates more precise imaging data with a significant increase in the resolution of intracellular structures containing BM proteins, as well as an enhanced signal-to-noise ratio. Moreover, this super-resolution microscopy technique is relatively easy to use. Since super-resolution imaging significantly increases resolution compared to confocal microscopy, up to 120 nm in the x- and y-axes and 350 nm in z-axis, this approach will greatly impact our understanding of the polarized deposition of BM by more accurately characterizing the intracellular trafficking and secretion of BM proteins in epithelial cells such as the FCs.
Moreover, another super-resolution imaging approach (Structured Illumination Microscopy, SIM) and the use of deconvolution algorithms designed for point-scanning confocal microscopy (i.e., Nikon&#39;s enhanced resolution software module) have been used previously to characterize the role of factors involved in BM deposition29. The increased resolution associated with these techniques have provided key insight into our understanding of the intracellular trafficking of BM proteins and the organization of the BM. However, these approaches present some limitations compared to Airyscan microscopy. For instance, SIM is not very efficient at acquiring optimal images when obtaining optical z-sections deep in the tissue. This makes SIM difficult to use when screening for new components involved in BM deposition. In addition, the use of deconvolution algorithms applied to confocal images does not achieve the same level of super-resolution. Overall, Airyscan super-resolution imaging on fixed tissue is a powerful and superior approach to study BM intracellular trafficking and deposition.
However, as with any other super-resolution microscopy, a few inconveniences are also associated with this approach, and need to be taken into account while imaging. First, the super-resolution mode usually requires longer acquisition time of the sample than confocal mode. Thus, the acquisition parameters and the region of interest have to be set carefully to minimize photobleaching the sample. Moreover, image files generated by super-resolution microscopy are much larger than files generated by confocal microscopy and may need specialized computers or servers for image processing and storage.
Finally, live imaging of the BM proteins during Drosophila oogenesis has been used to characterize new factors involved in BM polarity57. However, this approach has limitations, specifically in the resolution of intracellular structures during BM protein trafficking. Super-resolution microscopy is a powerful approach to use in conjunction with live imaging to precisely determine the roles of identified factors in the polarized deposition of BM proteins.
Other applications of this method to study the localization of intracellular components dedicated to vesicular trafficking using endogenously tagged proteins and super-resolution imaging 
The establishment and maintenance of tissues and organs during development and in an adult organism rely in part on cell-to-cell signaling and cellular tension and adhesion. These processes depend on intracellular trafficking, endocytosis, exocytosis, and secretion of specific proteins. Thus, the methods described herein to decipher the intracellular trafficking of BM using endogenously tagged proteins and confocal and super-resolution microscopy could be easily adapted to study each process. Moreover, the ease of use of CRISPR/Cas9-mediated genetic editing to generate endogenously tagged proteins make this approach even more versatile and powerful58.
In conclusion, we have described methods for the preparation and imaging of BM proteins in the FE of the Drosophila ovary using confocal and super-resolution microscopy. These protocols can be applied for high-throughput screening to identify new components dedicated to the proper placement of BM proteins. Finally, the employment of this methodology has the potential to make significant contributions to our understanding of how epithelial cells control the polarized secretion of BM proteins, a key process in the establishment and maintenance of epithelial architecture.

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><tbody><tr><td>Alexa Fluor 546 phalloidin</td><td>Invitrogen</td><td>A22283</td><td>F-Actin Stain (1/500 of 66&amp;micro;M)</td></tr><tr><td>Alexa Fluor 647 phalloidin</td><td>Invitrogen</td><td>A22287</td><td>F-Actin Stain (1/100 of 66&amp;micro;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&amp;micro;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 &lt;em&gt;Drosophila &lt;/em&gt;line (ZCL1700)</td><td></td><td></td><td>Morin et al., 2001</td></tr><tr><td>Fly Stock: UAS-&lt;em&gt;Crag &lt;/em&gt;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 &amp;micro;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&amp;nbsp; 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.

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

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

2.1K Views.  Northern Illinois University. Dit protocol maakt de karakterisering van de intracellulaire handel en secretie van keldermembraaneiwitten mogelijk met behulp van de Drosophila-array als modelsysteem. Het belangrijkste voordeel van onze techniek is dat het hoge resolutie beeldvorming van de intracellulaire handel van keldermembraaneiwitten mogelijk maakt met behulp van endogene eiwitten en Airyscan superresolutiemicroscopie. Hoewel dit protocol is ontwikkeld om de intracellulaire handel van keldermembraaneiwitten in beeld t...

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

Upright Imaging of Drosophila Egg Chambers

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in architecture, is well-characterized, and is amenable to genetic analysis. Each egg chamber consists of germ-line cells surrounded by a single epithelial layer of somatic follicle cells. Subsets of follicle cells undergo differentiation during specific stages to become several different cell types. Standard techniques primarily allow for a lateral view of egg chambers, and therefore a limited view of follicle cell organization and identity. The upright imaging protocol describes a mounting technique that enables a novel, vertical view of egg chambers with a standard confocal microscope. Samples are first mounted between two layers of glycerin jelly in a lateral (horizontal) position on a glass microscope slide. The jelly with encased egg chambers is then cut into blocks, transferred to a coverslip, and flipped to position egg chambers upright. Mounted egg chambers can be imaged on either an upright or an inverted confocal microscope. This technique enables the study of follicle cell specification, organization, molecular markers, and egg development with new detail and from a new perspective.

Upright Imaging of Drosophila Egg Chambers

The upright imaging method described in this protocol allows for the detailed visualization of the poles of a developing Drosophila melanogaster egg. This end-on view provides a new perspective into the arrangements and morphologies of multiple cell types in the follicular epithelium.

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in ...

Nuclear Migration in the Drosophila Oocyte

Live cell imaging is particularly necessary to understand the cellular and molecular mechanisms that regulate organelle movements, cytoskeleton rearrangements, or polarity patterning within the cells. When studying oocyte nucleus positioning, live-imaging techniques are essential to capture the dynamic events of this process. The Drosophila egg chamber is a multicellular structure and an excellent model system to study this phenomenon because of its large size and availability of numerous genetic tools. During Drosophila mid-oogenesis, the nucleus migrates from a central position within the oocyte to adopt an asymmetric position mediated by microtubule-generated forces. This migration and positioning of the nucleus are necessary to determine the polarity axes of the embryo and the subsequent adult fly. One characteristic of this migration is that it occurs in three dimensions (3D), creating a necessity for live imaging. Thus, to study the mechanisms that regulate nuclear migration, we have developed a protocol to culture the dissected egg chambers and perform live imaging for 12 h by time-lapse acquisitions using spinning-disk confocal microscopy. Overall, our conditions allow us to preserve Drosophila egg chambers alive for a long period of time, thereby enabling the completion of nuclear migration to be visualized in a large number of samples in 3D.

Nuclear Migration in the Drosophila Oocyte

In Drosophila, the oocyte nucleus undergoes microtubule-dependent migration during oogenesis. Here, we describe a protocol that was developed to follow the migration by performing live imaging on egg chambers ex-vivo. Our procedure maintains egg chambers alive for 12 h to acquire multi-position 3D time-lapse movies using spinning-disk confocal microscopy.

Live cell imaging is particularly necessary to understand the cellular and molecular mechanisms that regulate organelle movements, cytoskeleton ...

Visualizing Cytoskeleton-Dependent Trafficking of Lipid-Containing Organelles in Drosophila Embryos

Early Drosophila embryos are large cells containing a vast array of conventional and embryo-specific organelles. During the first three hours of embryogenesis, these organelles undergo dramatic movements powered by actin-based cytoplasmic streaming and motor-driven trafficking along microtubules. The development of a multitude of small, organelle-specific fluorescent probes (FPs) makes it possible to visualize a wide range of different lipid-containing structures in any genotype, allowing live imaging without requiring a genetically encoded fluorophore. This protocol shows how to inject vital dyes and molecular probes into Drosophila embryos to monitor the trafficking of specific organelles by live imaging. This approach is demonstrated by labeling lipid droplets (LDs) and following their bulk movement by particle image velocimetry (PIV). This protocol provides a strategy amenable to the study of other organelles, including lysosomes, mitochondria, yolk vesicles, and the ER, and for tracking the motion of individual LDs along microtubules. Using commercially available dyes brings the benefits of separation into the violet/blue and far-red regions of the spectrum. By multiplex co-labeling of organelles and/or cytoskeletal elements via microinjection, all the genetic resources in Drosophila are available for trafficking studies without the need to introduce fluorescently tagged proteins. Unlike genetically encoded fluorophores, which have low quantum yields and bleach easily, many of the available dyes allow for rapid and simultaneous capture of several channels with high photon yields.

Visualizing Cytoskeleton-Dependent Trafficking of Lipid-Containing Organelles in Drosophila Embryos

In the early Drosophila embryo, many organelles are motile. In principle, they can be imaged live via specific fluorescent probes, but the eggshell prevents direct application to the embryo. This protocol describes how to introduce such probes via microinjection, and then analyze bulk organelle motion via particle image velocimetry.

Early Drosophila embryos are large cells containing a vast array of conventional and embryo-specific organelles. During the first three hours of ...

Using Expansion Microscopy to Physically Enlarge Whole-Mount Drosophila Embryos for Super-Resolution Imaging

The workhorse of developmental biology is the confocal microscope, which allows researchers to determine the three-dimensional localization of tagged molecules within complex biological samples. While traditional confocal microscopes allow one to resolve two adjacent fluorescent point sources located a few hundred nanometers apart, observing the finer details of subcellular biology requires the ability to resolve signals in the order of tens of nanometers. Numerous hardware-based methods for super-resolution microscopy have been developed to allow researchers to sidestep such resolution limits, although these methods require specialized microscopes that are not available to all researchers. An alternative method for increasing resolving power is to isotropically enlarge the sample itself through a process known as expansion microscopy (ExM), which was first described by the Boyden group in 2015. ExM is not a type of microscopy per se but is rather a method for swelling a sample while preserving the relative spatial organization of its constituent molecules. The expanded sample can then be observed at an effectively increased resolution using a traditional confocal microscope. Here, we describe a protocol for implementing ExM in whole-mount Drosophila embryos, which is used to examine the localization of Par-3, myosin II, and mitochondria within the surface epithelial cells. This protocol yields an approximately four-fold increase in sample size, allowing for the detection of subcellular details that are not visible with conventional confocal microscopy. As proof of principle, an anti-GFP antibody is used to distinguish distinct pools of myosin-GFP between adjacent cell cortices, and fluorescently labeled streptavidin is used to detect endogenous biotinylated molecules to reveal the fine details of the mitochondrial network architecture. This protocol utilizes common antibodies and reagents for fluorescence labeling, and it should be compatible with many existing immunofluorescence protocols.

Using Expansion Microscopy to Physically Enlarge Whole-Mount Drosophila Embryos for Super-Resolution Imaging

Here, a protocol for the implementation of expansion microscopy in early Drosophila embryos to achieve super-resolution imaging using a conventional laser-scanning confocal microscope is presented.

The workhorse of developmental biology is the confocal microscope, which allows researchers to determine the three-dimensional localization of tagged ...

Real-Time Imaging of Germline Stem Cells in Drosophila Ex Vivo Germaria Culture

Extended Live Imaging of Female Drosophila melanogaster Germline Stem Cell Niches

Live imaging methods allow the analysis of dynamic cellular processes in detail and in real-time. The Drosophila ovary represents an excellent model to explore the dynamics of a myriad of developmental processes, such as cell division, stemness, differentiation, migration, apoptosis, autophagy, cellular adhesion, etc., over time. Recently, we have implemented an extended ex vivo culture and live imaging of the female Drosophila GSC niche. Using a Drosophila line harboring a GFP::Par-1 transgene as an example, this method allows the visualization of the GSCs&#39; asymmetric division within their niche and the description of the changes in the spectrosome morphology along the cell cycle. Here, we present a detailed protocol for the ex vivo culture of Drosophila germaria, enabling prolonged visualization of the female GSC niche. Importantly, this protocol is broadly applicable to live imaging GSCs with multiple fluorescently tagged proteins of interest that are available in stock centers and/or in the Drosophila research community.

Here, we provide a detailed protocol to perform live imaging of the asymmetric division of germline stem cells (GSCs) in the Drosophila ovarian niche. We use a transgenic line that ubiquitously expresses a green fluorescent protein (GFP) fusion of the spectrosome protein Par-1.

Live imaging methods allow the analysis of dynamic cellular processes in detail and in real-time. The Drosophila ovary represents an excellent model to ...

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. ...

Neuroscience

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

The Drosophila oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila oocytes1,2.
The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis3,4. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent5 and provides a useful system for investigating cytoskeleton function at these stages.
Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup.

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

A protocol for live imaging of GFP-tagged proteins or autofluorescent structures in individual Drosophila oocytes is described.

The  Drosophila oocyte has been established as a versatile system for investigating  fundamental questions such as cytoskeletal function, cell ...

Biology

Imaging Through the Pupal Case of Drosophila melanogaster

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 &#181;m of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.

Imaging Through the Pupal Case of Drosophila melanogaster

This paper demonstrates the use of a fast scanning confocal microscope to image cell behavior directly through the puparium. By leaving the pupal case intact, this method allows observation and measurement of dynamic cell processes at a stage of Drosophila development that is difficult to study directly.

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled ...

Super-Resolution Live Cell Imaging of Subcellular Structures

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally been restricted to be used only on fixed samples or live cells outside of tissue labeled with strong fluorescent signal. Current super-resolution live cell imaging techniques require the use of special fluorescence probes, high illumination, multiple image acquisitions with post-acquisition processing, or often a combination of these processes. These prerequisites significantly limit the biological samples and contexts that this technique can be applied to.
Here we describe a method to perform super-resolution (~140 nm XY-resolution) time-lapse fluorescence live cell imaging in situ. This technique is also compatible with low fluorescent intensity, for example, EGFP or mCherry endogenously tagged at lowly expressed genes. As a proof-of-principle, we have used this method to visualize multiple subcellular structures in the Drosophila testis. During tissue preparation, both the cellular structure and tissue morphology are maintained within the dissected testis. Here, we use this technique to image microtubule dynamics, the interactions between microtubules and the nuclear membrane, as well as the attachment of microtubules to centromeres.
This technique requires special procedures in sample preparation, sample mounting and immobilizing of specimens. Additionally, the specimens must be maintained for several hours after dissection without compromising cellular function and activity. While we have optimized the conditions for live super-resolution imaging specifically in Drosophila male germline stem cells (GSCs) and progenitor germ cells in dissected testis tissue, this technique is broadly applicable to a variety of different cell types. The ability to observe cells under their physiological conditions without sacrificing either spatial or temporal resolution will serve as an invaluable tool to researchers seeking to address crucial questions in cell biology.

Presented here is a protocol for super-resolution live-cell imaging in intact tissue. We have standardized the conditions for imaging a highly sensitive adult stem cell population in its native tissue environment. This technique involves balancing temporal and spatial resolution to allow for the direct observation of biological phenomena in live tissue.

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally ...

Studying Membrane Protein Trafficking in Drosophila Photoreceptor Cells Using eGFP-Tagged Proteins

Membrane protein trafficking regulates the incorporation and removal of receptors and ion channels into the plasma membrane. This process is fundamentally important for cell function and cell integrity of neurons. Drosophila photoreceptor cells have become a model for studying membrane protein trafficking. Besides rhodopsin, which upon illumination becomes internalized from the photoreceptor membrane and is degraded, the transient receptor potential-like (TRPL) ion channel in Drosophila exhibits a light-dependent translocation between the rhabdomeral photoreceptor membrane (where it is located in the dark) and the photoreceptor cell body (to which it is transported upon illumination). This intracellular transport of TRPL can be studied in a simple and non-invasive way by expressing eGFP-tagged TRPL in photoreceptor cells. The eGFP fluorescence can then be observed either in the deep pseudopupil or by water immersion microscopy. These methods allow detection of fluorescence in the intact eye and are therefore useful for high-throughput assays and genetic screens for Drosophila mutants defective in TRPL translocation. Here, the preparation of flies, the microscopic techniques, as well as quantification methods used to study this light-triggered translocation of TRPL are explained in detail. These methods can be applied also for trafficking studies on other Drosophila photoreceptor proteins, for example, rhodopsin. In addition, by using eGFP-tagged rhabdomeral proteins, these methods can be used to assess the degeneration of photoreceptor cells.

Studying Membrane Protein Trafficking in Drosophila Photoreceptor Cells Using eGFP-Tagged Proteins

Here, non-invasive methods are described for localization of photoreceptor membrane proteins and assessment of retinal degeneration in the Drosophila compound eye using eGFP fluorescence.

Membrane protein trafficking regulates the incorporation and removal of receptors and ion channels into the plasma membrane. This process is fundamentally ...

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

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