Cell-type-specific Labeling of Endogenous Proteins Using the Split GFP System in Drosophila.

Accurate identification of the locations of endogenous proteins is crucial for understanding their functions in tissues and cells. However, achieving precise cell-type-specific labeling of these proteins has been challenging in vivo. A notable solution to this challenge is the self-complementing split green fluorescent protein (GFP1-10/11) system. In this paper, we present a detailed protocol for labeling endogenous proteins in a cell-type-specific manner using the GFP1-10/11 system in fruit flies. This approach depends on the automatic reconstitution of the GFP1-10 and GFP11 fragments, creating a fluorescence signal. We insert the GFP11 fragment into a specific genomic locus while expressing its counterpart, GFP1-10, through an available Gal4 driver line. The unique aspect of this system is that neither GFP1-10 nor GFP11 alone emits fluorescence, enabling the precise detection of protein localization only in Gal4-positive cells. We illustrate this technique using the adhesion molecule gene teneurin-m (Ten-m) as a model, highlighting the generation and validation of GFP11 protein trap lines via Minos-mediated integration cassette (MiMIC) insertion. Furthermore, we demonstrate the cell-type-specific labeling of Ten-m proteins in the larval brains of fruit flies. This method significantly enhances our ability to image endogenous protein localization patterns in a cell-type-specific manner and is adaptable to various model organisms beyond fruit flies.

Adjacent Neuronal Fascicle Guides Motoneuron 24 Dendritic Branching and Axonal Routing Decisions through Dscam1 Signaling.

The formation and precise positioning of axons and dendrites are crucial for the development of neural circuits. Although juxtracrine signaling via cell-cell contact is known to influence these processes, the specific structures and mechanisms regulating neuronal process positioning within the central nervous system (CNS) remain to be fully identified. Our study investigates motoneuron 24 (MN24) in the Drosophila embryonic CNS, which is characterized by a complex yet stereotyped axon projection pattern, known as 'axonal routing.' In this motoneuron, the primary dendritic branches project laterally toward the midline, specifically emerging at the sites where axons turn. We observed that Scp2-positive neurons contribute to the lateral fascicle structure in the ventral nerve cord (VNC) near MN24 dendrites. Notably, the knockout of the Down syndrome cell adhesion molecule (dscam1) results in the loss of dendrites and disruption of proper axonal routing in MN24, while not affecting the formation of the fascicle structure. Through cell-type specific knockdown and rescue experiments of dscam1, we have determined that the interaction between MN24 and Scp2-positive fascicle, mediated by Dscam1, promotes the development of both dendrites and axonal routing. Our findings demonstrate that the holistic configuration of neuronal structures, such as axons and dendrites, within single motoneurons can be governed by local contact with the adjacent neuron fascicle, a novel reference structure for neural circuitry wiring.

CRISPR/Cas9-mediated knock-in in ebony gene using a PCR product donor template in Drosophila.

CRISPR/Cas9 technology has been a powerful tool for gene editing in Drosophila, particularly for knocking in base-pair mutations or a variety of gene cassettes into endogenous gene loci. Among the Drosophila community, there has been a concerted effort to establish CRISPR/Cas9-mediated knock-in protocols that decrease the amount of time spent on molecular cloning. Here, we report the CRISPR/Cas9-mediated insertion of a ~50 base-pair sequence into the ebony gene locus, using a linear double-stranded DNA (PCR product) donor template By circumventing the cloning step of the donor template, our approach suggests the PCR product as a useful alternative knock-in donor format.

CRISPR-Cas9-Mediated Knock-In Approach to Insert the GFP11 Tag into the Genome of a Human Cell Line.

The protocol in this chapter describes a method to label endogenous proteins using a self-complementing split green fluorescent protein (split GFP1-10/11) in a human cell line. By directly delivering Cas9/sgRNA ribonucleoprotein (RNP) complexes through nucleofection, this protocol allows for the efficient integration of GFP11 into a specific genomic locus via CRISPR-Cas9-mediated homology-directed repair (HDR). We use the GFP11 sequence in the form of a single-stranded DNA (ssDNA) as an HDR template. Because the ssDNA with less than 200 nucleotides used here is commercially synthesized, this approach remains cloning-free. The integration of GFP11 is performed in cells stably expressing GFP1-10, thereby inducing fluorescence reconstitution. Subsequently, such a reconstituted signal is analyzed using fluorescence flow cytometry for estimating knock-in efficiencies and enriching the GFP-positive cell population. Finally, the enriched cells can be visualized using fluorescence microscopy.

A Genetic Toolkit for Simultaneous Generation of LexA- and QF-Expressing Clones in Selected Cell Types in Drosophila.

Visualization and manipulation of defined motoneurons have provided significant insights into how motor circuits are assembled in Drosophila. A conventional approach for molecular and cellular analyses of subsets of motoneurons involves the expression of a wide range of UAS transgenes using available GAL4 drivers (eg, eve promoter-fused GAL4). However, a more powerful toolkit could be one that enables a single-cell characterization of interactions between neurites from neurons of interest. Here we show the development of a UAS > LexA > QF expression system to generate randomly selected neurons expressing one of the 2 binary expression systems. As a demonstration, we apply it to visualize dendrite-dendrite interactions by genetically labeling eve + neurons with distinct fluorescent reporters.

Imaging of In Vitro and In Vivo Neurons in Drosophila Using Stochastic Optical Reconstruction Microscopy.

The Drosophila melanogaster brain comprises different neuronal cell types that interconnect with precise patterns of synaptic connections. These patterns are essential for the normal function of the brain. To understand the connectivity patterns requires characterizing them at single-cell resolution, for which a fluorescence microscope becomes an indispensable tool. Additionally, because the neurons connect at the nanoscale, the investigation often demands super-resolution microscopy. Here, we adopt one super-resolution microscopy technique, called stochastic optical reconstruction microscopy (STORM), improving the lateral and axial resolution to ∼20 nm. This article extensively describes our methods along with considerations for sample preparation of neurons in vitro and in vivo, conjugation of dyes to antibodies, immunofluorescence labeling, and acquisition and processing of STORM data. With these tools and techniques, we open up the potential to investigate cell-cell interactions using STORM in the Drosophila nervous system. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Preparation of Drosophila primary neuronal culture and embryonic fillets Basic Protocol 2: Immunofluorescence labeling of samples Basic Protocol 3: Single-molecule fluorescence imaging Basic Protocol 4: Localization and visualization of single-molecule data Supporting Protocol: Conjugation of antibodies with STORM-compatible dyes.

Cell-type-specific, multicolor labeling of endogenous proteins with split fluorescent protein tags in Drosophila.

The impact of the Drosophila experimental system on studies of modern biology cannot be understated. The ability to tag endogenously expressed proteins is essential to maximize the use of this model organism. Here, we describe a method for labeling endogenous proteins with self-complementing split fluorescent proteins (split FPs) in a cell-type-specific manner in Drosophila A short fragment of an FP coding sequence is inserted into a specific genomic locus while the remainder of the FP is expressed using an available GAL4 driver line. In consequence, complementation fluorescence allows examination of protein localization in particular cells. Besides, when inserting tandem repeats of the short FP fragment at the same genomic locus, we can substantially enhance the fluorescence signal. The enhanced signal is of great value in live-cell imaging at the subcellular level. We can also accomplish a multicolor labeling system with orthogonal split FPs. However, other orthogonal split FPs do not function for in vivo imaging besides split GFP. Through protein engineering and in vivo functional studies, we report a red split FP that we can use for duplexed visualization of endogenous proteins in intricate Drosophila tissues. Using the two orthogonal split FP systems, we have simultaneously imaged proteins that reside in distinct subsynaptic compartments. Our approach allows us to study the proximity between and localization of multiple proteins endogenously expressed in essentially any cell type in Drosophila.

Multiplexed labeling of cellular proteins with split fluorescent protein tags.

Self-complementing split fluorescent proteins (split FP1-10/11) have become an important labeling tool in live-cell protein imaging. However, current split FP systems to label multiple proteins in single cells have a fundamental limitation in the number of proteins that can be simultaneously labeled. Here, we describe an approach to expand the number of orthogonal split FP systems with spectrally distinct colors. By combining rational design and cycles of directed evolution, we expand the spectral color palette of FP1-10/11. We also circularly permutate GFP and synthesize the ß-strand 7, 8, or 10 system. These split GFP pairs are not only capable of labeling proteins but are also orthogonal to the current FP1-10/11 pairs, offering multiplexed labeling of cellular proteins. Our multiplexing approach, using the new orthogonal split FP systems, demonstrates simultaneous imaging of four distinct proteins in single cells; the resulting images reveal nuclear localization of focal adhesion protein Zyxin.

Retrograde Tracing of Drosophila Embryonic Motor Neurons Using Lipophilic Fluorescent Dyes.

We describe a technique for retrograde labeling of motor neurons in Drosophila. We use an oil-dissolved lipophilic dye and deliver a small droplet to an embryonic fillet preparation by a microinjector. Each motor neuron whose membrane is contacted by the droplet can then be rapidly labeled. Individual motor neurons are continuously labeled, enabling fine structural details to be clearly visualized. Given that lipophilic dyes come in various colors, the technique also provides a means to get adjacent neurons labeled in multicolor. This tracing technique is therefore useful for studying neuronal morphogenesis and synaptic connectivity in the motor neuron system of Drosophila.

Autoinhibition of kinesin-1 is essential to the dendrite-specific localization of Golgi outposts.

Neuronal polarity relies on the selective localization of cargo to axons or dendrites. The molecular motor kinesin-1 moves cargo into axons but is also active in dendrites. This raises the question of how kinesin-1 activity is regulated to maintain the compartment-specific localization of cargo. Our in vivo structure-function analysis of endogenous Drosophila melanogaster kinesin-1 reveals a novel role for autoinhibition in enabling the dendrite-specific localization of Golgi outposts. Mutations that disrupt kinesin-1 autoinhibition result in the axonal mislocalization of Golgi outposts. Autoinhibition also regulates kinesin-1 localization. Uninhibited kinesin-1 accumulates in axons and is depleted from dendrites, correlating with the change in outpost distribution and dendrite growth defects. Genetic interaction tests show that a balance of kinesin-1 inhibition and dynein activity is necessary to localize Golgi outposts to dendrites and keep them from entering axons. Our data indicate that kinesin-1 activity is precisely regulated by autoinhibition to achieve the selective localization of dendritic cargo.

A scalable strategy for high-throughput GFP tagging of endogenous human proteins.

A central challenge of the postgenomic era is to comprehensively characterize the cellular role of the ∼20,000 proteins encoded in the human genome. To systematically study protein function in a native cellular background, libraries of human cell lines expressing proteins tagged with a functional sequence at their endogenous loci would be very valuable. Here, using electroporation of Cas9 nuclease/single-guide RNA ribonucleoproteins and taking advantage of a split-GFP system, we describe a scalable method for the robust, scarless, and specific tagging of endogenous human genes with GFP. Our approach requires no molecular cloning and allows a large number of cell lines to be processed in parallel. We demonstrate the scalability of our method by targeting 48 human genes and show that the resulting GFP fluorescence correlates with protein expression levels. We next present how our protocols can be easily adapted for the tagging of a given target with GFP repeats, critically enabling the study of low-abundance proteins. Finally, we show that our GFP tagging approach allows the biochemical isolation of native protein complexes for proteomic studies. Taken together, our results pave the way for the large-scale generation of endogenously tagged human cell lines for the proteome-wide analysis of protein localization and interaction networks in a native cellular context.

Versatile protein tagging in cells with split fluorescent protein.

In addition to the popular method of fluorescent protein fusion, live cell protein imaging has now seen more and more application of epitope tags. The small size of these tags may reduce functional perturbation and enable signal amplification. To address their background issue, we adapt self-complementing split fluorescent proteins as epitope tags for live cell protein labelling. The two tags, GFP11 and sfCherry11 are derived from the eleventh ß-strand of super-folder GFP and sfCherry, respectively. The small size of FP11-tags enables a cost-effective and scalable way to insert them into endogenous genomic loci via CRISPR-mediated homology-directed repair. Tandem arrangement FP11-tags allows proportional enhancement of fluorescence signal in tracking intraflagellar transport particles, or reduction of photobleaching for live microtubule imaging. Finally, we show the utility of tandem GFP11-tag in scaffolding protein oligomerization. These experiments illustrate the versatility of FP11-tag as a labelling tool as well as a multimerization-control tool for both imaging and non-imaging applications.

Specification of Dendritogenesis Site in Drosophila aCC Motoneuron by Membrane Enrichment of Pak1 through Dscam1.

Precise positioning of dendritic branches is a critical step in the establishment of neuronal circuitry. However, there is limited knowledge on how environmental cues translate into dendrite initiation or branching at a specific position. Here, through a combination of mutation, RNAi, and imaging experiments, we found that a Dscam-Dock-Pak1 hierarchical interaction defines the stereotypical dendrite growth site in the Drosophila aCC motoneuron. This interaction localizes the Cdc42 effector Pak1 to the plasma membrane at the dendrite initiation site before the activation of Cdc42. Ectopic expression of membrane-anchored Pak1 overrides this spatial specification of dendritogenesis, confirming its function in guiding Cdc42 signaling. We further discovered that Dscam1 localization in aCC occurs through an inter-neuronal contact that involves Dscam1 in the partner MP1 neuron. These findings elucidate a mechanism by which Dscam1 controls neuronal morphogenesis through spatial regulation of Cdc42 signaling and, subsequently, cytoskeletal remodeling.

Heavy water: a simple solution to increasing the brightness of fluorescent proteins in super-resolution imaging.

Photoactivatable fluorescent proteins (PA-FPs) are widely used in live single-molecule super-resolution imaging but emit substantially fewer photons than organic dyes do. Herein, we show that in heavy water (D2O) instead of H2O, common PA-FPs emit 26-54% more photons, effectively improving the localization precision in super-resolution imaging.

Open-top selective plane illumination microscope for conventionally mounted specimens.

We have developed a new open-top selective plane illumination microscope (SPIM) compatible with microfluidic devices, multi-well plates, and other sample formats used in conventional inverted microscopy. Its key element is a water prism that compensates for the aberrations introduced when imaging at 45 degrees through a coverglass. We have demonstrated its unique high-content imaging capability by recording Drosophila embryo development in environmentally-controlled microfluidic channels and imaging zebrafish embryos in 96-well plates. We have also shown the imaging of C. elegans and moving Drosophila larvae on coverslips.

Imaging dynamic molecular signaling by the Cdc42 GTPase within the developing CNS.

Protein interactions underlie the complexity of neuronal function. Potential interactions between specific proteins in the brain are predicted from assays based on genetic interaction and/or biochemistry. Genetic interaction reveals endogenous, but not necessarily direct, interactions between the proteins. Biochemistry-based assays, on the other hand, demonstrate direct interactions between proteins, but often outside their native environment or without a subcellular context. We aimed to achieve the best of both approaches by visualizing protein interaction directly within the brain of a live animal. Here, we show a proof-of-principle experiment in which the Cdc42 GTPase associates with its alleged partner WASp within neurons during the time and space that coincide with the newly developing CNS.

Active Microscope Stabilization in Three Dimensions Using Image Correlation.

BACKGROUND: Super-resolution microscopy techniques are often extremely susceptible to sample drift due to their high spatial resolution and the long time needed for data acquisition. While several techniques for stabilizing against drift exist, many require complicated additional hardware or intrusive sample preparations. We introduce a method that requires no additional sample preparation, is simple to implement and simultaneously corrects for x, y and z drift. RESULTS: We use bright-field images of the specimen itself to calculate drift in all three dimensions: x, y and z. Bright-field images are acquired on an inexpensive CCD. By correlating each acquired bright-field image with an in-focus and two out-of-focus reference images we determine and actively correct for drift at rates of a few Hertz. This method can maintain stability to within 10 nm for x and y and 20 nm for z over several minutes. CONCLUSION: Our active drift stabilization system is capable of simultaneously compensating x, y and z drift through an image-based correlation method that requires no special sample treatment or extensive microscope modifications. While other techniques may provide better stability, especially for higher frequency drift, our method is easy to implement and widely applicable in terms of both sample type and microscopy technique.

Development in the STORM.

The recent invention of superresolution microscopy has brought up much excitement in the biological research community. Here, we focus on stochastic optical reconstruction microscopy/photoactivated localization microscopy (STORM/PALM) to discuss the challenges in applying superresolution microscopy to the study of developmental biology, including tissue imaging, sample preparation artifacts, and image interpretation. We also summarize new opportunities that superresolution microscopy could bring to the field of developmental biology.

Endogenous activation patterns of Cdc42 GTPase within Drosophila embryos.

Knowing when and where a given protein is activated within intact animals assists in elucidating its in vivo function. With the use of a genetically encoded A-probe (activation bioprobe), we revealed that Cdc42 guanosine triphosphatase (GTPase) remains inactive within Drosophila embryos during the first two-thirds of embryogenesis. Within the central nervous system where Cdc42 activity first becomes up-regulated, individual neurons display patterns restricted to specific subcellular compartments. At both organismal and cellular levels, Cdc42's endogenous activation patterns in the wild type allow predictions of where loss-of-function phenotypes will emerge in cdc42/cdc42 mutants. Genetic tests support the importance of suppressing endogenous Cdc42 activities until needed. Thus, bioprobe-assisted analysis uncovers how ubiquitously expressed signaling proteins control cellular events through continual regulation of their activities within animals.

Rapid frequency-domain FLIM spinning disk confocal microscope: lifetime resolution, image improvement and wavelet analysis.

A spinning disk confocal attachment is added to a full-field real-time frequency-domain fluorescence lifetime-resolved imaging microscope (FLIM). This provides confocal 3-D imaging while retaining all the characteristics of the normal 2-D FLIM. The spinning disk arrangement allows us to retain the speed of the normal 2-D full field FLIM while gaining true 3-D resolution. We also introduce the use of wavelet image transformations into the FLIM analysis. Wavelets prove useful for selecting objects according to their morphology, denoising and background subtraction. The performance of the instrument and the analysis routines are tested with quantitative physical samples and examples are presented with complex biological samples.