Daichi Kamiyama

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 β-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.

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

Significance The function of a large fraction of the human proteome still remains poorly characterized. Tagging proteins with a functional sequence is a powerful way to access function, and inserting tags at endogenous genomic loci allows the preservation of a near-native cellular background. To characterize the cellular role of human proteins in a systematic manner and in a native context, we developed a method for tagging endogenous human proteins with GFP that is both rapid and readily applicable at a genome-wide scale. Our approach allows studying both localization and interaction partners of the protein target. Our results pave the way for the large-scale generation of endogenously tagged human cell lines for a systematic functional interrogation of the human proteome. 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.

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.

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.

Active microscope stabilization in three dimensions using image correlation

BackgroundSuper-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.ResultsWe 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.ConclusionOur 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.

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.

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.

Open-top selective plane illumination microscope compatible with standard sample holders

Selective plane illumination microscopy (SPIM) has emerged recently as a powerful tool in optically sectioning tissue samples. We have designed a new SPIM that allows samples to be mounted in a variety of common methods.