With recent advances in fluorescence microscopy, resolution is often limited by the size of the label and the resulting linkage error, rather than the microscope itself. Site-specific incorporation of noncanonical amino acids (ncAAs) combined with bioorthogonal click chemistry provides a powerful tool for fluorescent protein labeling, overcoming the spatial uncertainty inherent to antibody-based probes. Here, we present a method to further improve labeling precision by combining ncAA labeling with expansion microscopy (ExM) for dual-color super-resolution imaging. After optimizing labeling procedures and fluorophore selection, we visualize and resolve the nanoscale distribution of Na,K-ATPase α1 and β1 subunits in expanded HEK 293T cells. We validate our approach by super-resolution STED imaging of the ncAA labeled β1 subunit in unexpanded cells. This work presents a strong framework for multiplexed, high-resolution imaging, suggesting that ncAA labeling combined with ExM enables biological imaging at the nanometer scale

Rubisco is the main entry point of inorganic carbon into the biosphere and a central player in the global carbon system. The relatively low specific activity and tendency to accept O2 as a substrate have made Rubisco an attractive but challenging target for enzyme engineering. We have developed an enzyme engineering and screening platform for Rubisco using the model cyanobacterium Synechocystis sp. PCC 6803. Starting with the Form II Rubisco from Gallionella, we first show that the enzyme can replace the native Form I Rubisco in Synechocystis and that growth rates become sensitive to CO2 and O2 levels. We address the challenge of designing a zero-shot input library of the Gallionella Rubisco, without prior experimental knowledge, by coupling the phylogenetically guided model EV mutation with "in silico evolution". This multisite mutagenesis library of Synechocystis (n = 16) was subjected to competitive growth in different gas feeds coupled to deep sequencing, in order to compare Rubisco variants. We identified an amino acid exchange that increased the thermostability of Gallionella Rubisco and conveyed resilience to otherwise detrimental amino acid exchanges. The platform is a first step toward high-throughput screening of Rubisco variants in Synechocystis and creating optimized enzyme variants to accelerate the Calvin-Benson-Bassham cycle in cyanobacteria and possibly chloroplasts.

Mutations in the gene encoding the alpha3 Na+/K+-ATPase isoform (ATP1A3) lead to movement disorders that manifest with dystonia, a common neurological symptom with many different origins, but for which the underlying molecular mechanisms remain poorly understood. We have generated an ATP1A3 mutant mouse that displays motor impairments and a hyperexcitable motor phenotype compatible with dystonia. We show that neurons harbouring this mutation are compromised in their ability to extrude raised levels of intracellular sodium, highlighting a profound deficit in neuronal sodium homeostasis. We show that the spinal motor network in ATP1A3 mutant mice has a reduced responsiveness to activity-dependent rises in intracellular sodium and that this is accompanied by loss of the Na+/K+-ATPase-mediated afterhyperpolarization in motor neurons. Taken together, our data support that the alpha3 Na+/K+-ATPase is important for cellular and spinal motor network homeostasis. These insights suggest that it may be useful to consider ways to compensate for this loss of a critical afterhyperpolarization-dependent control of neuronal excitability when developing future therapies for dystonia.

DNA origami-based nanotechnology is a versatile tool for exploring fundamental biological questions and holds significant promise for future biomedical applications. Here, we leverage the optical transparency of the embryonic zebrafish to analyze live embryos injected intravenously with fluorescently labeled wireframe DNA origami nanosheets. Our approach integrated long-term, high-resolution imaging of transgenic live zebrafish embryos with single-cell RNA sequencing to elucidate the effects of oligolysine-polyethylene glycol copolymer (K-PEG) coating on the biodistribution of fluorescence signal in embryos injected with wireframe DNA origami nanosheets. We observed rapid accumulation of fluorescence signal in the caudal hematopoietic tissue (CHT). K-PEG coating mitigated the accumulation of fluorescence signal in CHT, enabling increased detection of signal in other tissues. Our findings highlighted the pivotal role of scavenger endothelial cells in DNA origami clearance, with K-PEG enabling the prolonged detection of fluorescence signal at the CHT. Furthermore, using a transgenic zebrafish line designed for targeted macrophage ablation, we found that macrophages contribute to the clearance of fluorescence signal in embryos injected with the noncoated but not with K-PEG-coated nanosheets. This study introduces a framework for the analyses of the biodistribution and clearance of DNA origami nanostructures in vivo with single-cell resolution in zebrafish models.

The emerging strategy of protein–drug conjugates (PDCs) for targeted cancer therapy holds great potential to improve treatment efficacy by specifically targeting cancer biomarkers and delivering toxic payloads directly to tumor cells, minimizing off-target toxicity. The success of this approach depends on the internalization and retention of the payload in target cells. This study introduces a method using a small protein domain engineered for conditional target affinity, enabling lysosomal trafficking independent of the biological fate of the receptor. Specifically, we describe the development of an EGF receptor binder, CaRAEGFR, with calcium-regulated affinity (CaRA), meaning the target binding strength is tailored by the available calcium concentration. This allows for endosomal dissociation, as calcium levels are lower in endosomes than in the bloodstream. Affinity measurements and structural modeling reveal the molecular basis of the calcium modulated affinity. Live cell imaging demonstrates efficient internalization and lysosomal trafficking of the calcium-dependent domain, while the EGF receptor is recycled to the membrane. When used as a drug carrier, CaRAEGFR effectively delivers the toxin to the lysosomes, resulting in potent cytotoxicity with an IC50 of 0.8 nM in EGFR-expressing cancer cells

Correct chromosome segregation is essential to preserve genetic integrity. The two protein kinases, Aurora B and its meiotic homolog Aurora C, regulate attachments between chromosomal kinetochores and microtubules, thereby contributing to the accuracy of the chromosome segregation process. Here we performed a detailed examination of the localization and activity of Aurora B/C kinases, their partner Incenp and the kinetochore target Hec1, during the second meiotic division in mouse oocytes. We found that a majority of Aurora B and C changed their localization from the outer kinetochore region of chromosomes at prometaphase II to an inner central region localized between sister centromeres at metaphase II. Depletion of the Aurora B/C pool at the inner central region using the haspin kinase inhibitor 5-iodotubercidin resulted in chromosome misalignments at the metaphase II stage. To further understand the role of the Aurora B/C pool at the central region, we examined the behaviour of single chromatids, that lack a central Aurora B/C pool but retain Aurora B/C at the outer kinetochores. We found that kinetochore-microtubule attachments at single chromatids were corrected at both prometaphase II and metaphase II stages, but that single chromatids compared to paired chromatids were more prone to misalignments following treatment of oocytes with the Aurora B/C inhibitory drugs AZD1152 and GSK1070916. We conclude that the Aurora B/C pool at the inner central region stabilizes chromosome alignment during metaphase II arrest, while Aurora B/C localized at the kinetochore assist in re-establishing chromosome positioning at the metaphase plate if alignment is lost. Collaboratively these two pools prevent missegregation and aneuploidy at the second meiotic division in mammalian oocytes.

Despite widespread adoption of tissue clearing techniques in recent years, poor access to suitable light-sheet fluorescence microscopes remains a major obstacle for biomedical end-users. Here, we present descSPIM (desktop-equipped SPIM for cleared specimens), a low-cost ($20,000–50,000), low-expertise (one-day installation by a non-expert), yet practical do-it-yourself light-sheet microscope as a solution for this bottleneck. Even the most fundamental configuration of descSPIM enables multi-color imaging of whole mouse brains and a cancer cell line-derived xenograft tumor mass for the visualization of neurocircuitry, assessment of drug distribution, and pathological examination by false-colored hematoxylin and eosin staining in a three-dimensional manner. Academically open-sourced (https://github.com/dbsb-juntendo/descSPIM), descSPIM allows routine three-dimensional imaging of cleared samples in minutes. Thus, the dissemination of descSPIM will accelerate biomedical discoveries driven by tissue clearing technologies.

The sodium potassium pump, Na,K-ATPase (NKA), is an integral plasma membrane protein, expressed in all eukaryotic cells. It is responsible for maintaining the transmembrane Na+ gradient and is the major determinant of the membrane potential. Self-interaction and oligomerization of NKA in cell membranes has been proposed and discussed but is still an open question. Here, we have used a combination of FRET and Fluorescence Correlation Spectroscopy, FRET-FCS, to analyze NKA in the plasma membrane of living cells. Click chemistry was used to conjugate the fluorescent labels Alexa 488 and Alexa 647 to non-canonical amino acids introduced in the NKA α1 and β1 subunits. We demonstrate that FRET-FCS can detect an order of magnitude lower concentration of green-red labeled protein pairs in a single-labeled red and green background than what is possible with cross-correlation (FCCS). We show that a significant fraction of NKA is expressed as a dimer in the plasma membrane. We also introduce a method to estimate not only the number of single and double labeled NKA, but the number of unlabeled, endogenous NKA and estimate the density of endogenous NKA at the plasma membrane to 1400 ± 800 enzymes/μm2.

Studying the nanoscale dynamics of subcellular structures is possible with 2D structured illumination microscopy (SIM). The method allows for acquisition with improved resolution over typical widefield. For 3D samples, the acquisition speed is inherently limited by the need to acquire sequential two-dimensional planes to create a volume. Here, we present a development of multifocus SIM designed to provide high volumetric frame rate by using fast synchronized electro-optical components. We demonstrate the high volumetric imaging capacity of the microscope by recording the dynamics of microtubule and endoplasmatic reticulum in living cells at up to 2.3 super resolution volumes per second for a total volume of 30 × 30 × 1.8 µm3,.

Membrane contact sites (MCSs) are hubs allowing various cell organelles to coordinate their activities. The dynamic nature of these sites and their small size hinder analysis by current imaging techniques. To overcome these limitations, we here design a series of reversible chemogenetic reporters incorporating improved, low-affinity variants of splitFAST, and study the dynamics of different MCSs at high spatiotemporal resolution, both in vitro and in vivo. We demonstrate that these versatile reporters suit different experimental setups well, allowing one to address challenging biological questions. Using these probes, we identify a pathway in which calcium (Ca²⁺) signalling dynamically regulates endoplasmic reticulum-mitochondria juxtaposition, characterizing the underlying mechanism. Finally, by integrating Ca²⁺-sensing capabilities into the splitFAST technology, we introduce PRINCESS (PRobe for INterorganelle Ca²⁺-Exchange Sites based on SplitFAST), a class of reporters to simultaneously detect MCSs and measure the associated Ca2+ dynamics using a single biosensor.