Prion-like protein aggregates exploit the RHO GTPase to cofilin-1 signaling pathway to enter cells.

Protein aggregation is a hallmark of diverse neurodegenerative diseases. Multiple lines of evidence have revealed that protein aggregates can penetrate inside cells and spread like prions. How such aggregates enter cells remains elusive. Through a focused siRNA screen targeting genes involved in membrane trafficking, we discovered that mutant SOD1 aggregates, like viruses, exploit cofilin-1 to remodel cortical actin and enter cells. Upstream of cofilin-1, signalling from the RHO GTPase and the ROCK1 and LIMK1 kinases controls cofilin-1 activity to remodel actin and modulate aggregate entry. In the spinal cord of symptomatic SOD1G93A transgenic mice, cofilin-1 phosphorylation is increased and actin dynamics altered. Importantly, the RHO to cofilin-1 signalling pathway also modulates entry of tau and α-synuclein aggregates. Our results identify a common host cell signalling pathway that diverse protein aggregates exploit to remodel actin and enter cells.

Genetic code expansion in the mouse brain.

Site-specific incorporation of non-natural amino acids into proteins, via genetic code expansion with pyrrolysyl tRNA synthetase (PylRS) and tRNA(Pyl)CUA pairs (and their evolved derivatives) from Methanosarcina sp., forms the basis of powerful approaches to probe and control protein function in cells and invertebrate organisms. Here we demonstrate that adeno-associated viral delivery of these pairs enables efficient genetic code expansion in primary neuronal culture, organotypic brain slices and the brains of live mice.

Memory Retrieval Has a Dynamic Influence on the Maintenance Mechanisms That Are Sensitive to ζ-Inhibitory Peptide (ZIP).

In neuroscientists' attempts to understand the long-term storage of memory, topics of particular importance and interest are the cellular and system mechanisms of maintenance (e.g., those sensitive to ζ-inhibitory peptide, ZIP) and those induced by memory retrieval (i.e., reconsolidation). Much is known about each of these processes in isolation, but less is known concerning how they interact. It is known that ZIP sensitivity and memory retrieval share at least some molecular targets (e.g., recycling α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, AMPA, receptors to the plasma membrane); conversely, the fact that sensitivity to ZIP emerges only after consolidation ends suggests that consolidation (and by extension reconsolidation) and maintenance might be mutually exclusive processes, the onset of one canceling the other. Here, we use conditioned taste aversion (CTA) in rats, a cortically dependent learning paradigm, to test this hypothesis. First, we demonstrate that ZIP infusions into gustatory cortex begin interfering with CTA memory 43-45 h after memory acquisition-after consolidation ends. Next, we show that a retrieval trial administered after this time point interrupts the ability of ZIP to induce amnesia and that ZIP's ability to induce amnesia is reengaged only 45 h after retrieval. This pattern of results suggests that memory retrieval and ZIP-sensitive maintenance mechanisms are mutually exclusive and that the progression from one to the other are similar after acquisition and retrieval. They also reveal concrete differences between ZIP-sensitive mechanisms induced by acquisition and retrieval: the latency with which ZIP-sensitive mechanisms are expressed differ for the two processes. SIGNIFICANCE STATEMENT: Memory retrieval and the molecular mechanisms that are sensitive to ζ-inhibitory peptide (ZIP) are the few manipulations that have been shown to effect memory maintenance. Although much is known about their effect on maintenance separately, it is unknown how they interact. Here, we describe a model for the interaction between memory retrieval and ZIP-sensitive mechanisms, showing that retrieval trials briefly (i.e., for 45 h) interrupt these mechanisms. ZIP sensitivity emerges across a similar time window after memory acquisition and retrieval; the maintenance mechanisms that follow acquisition and retrieval differ, however, in the latency with which the impact of ZIP is expressed.

Genetic code expansion enables live-cell and super-resolution imaging of site-specifically labeled cellular proteins.

Methods to site-specifically and densely label proteins in cellular ultrastructures with small, bright, and photostable fluorophores would substantially advance super-resolution imaging. Recent advances in genetic code expansion and bioorthogonal chemistry have enabled the site-specific labeling of proteins. However, the efficient incorporation of unnatural amino acids into proteins and the specific, fluorescent labeling of the intracellular ultrastructures they form for subdiffraction imaging has not been accomplished. Two challenges have limited progress in this area: (i) the low efficiency of unnatural amino acid incorporation that limits labeling density and therefore spatial resolution and (ii) the uncharacterized specificity of intracellular labeling that will define signal-to-noise, and ultimately resolution, in imaging. Here we demonstrate the efficient production of cystoskeletal proteins (ß-actin and vimentin) containing bicyclo[6.1.0]nonyne-lysine at genetically defined sites. We demonstrate their selective fluorescent labeling with respect to the proteome of living cells using tetrazine-fluorophore conjugates, creating densely labeled cytoskeletal ultrastructures. STORM imaging of these densely labeled ultrastructures reveals subdiffraction features, including nuclear actin filaments. This work enables the site-specific, live-cell, fluorescent labeling of intracellular proteins at high density for super-resolution imaging of ultrastructural features within cells.

Continuous polo-like kinase 1 activity regulates diffusion to maintain centrosome self-organization during mitosis.

Whether mitotic structures like the centrosome can self-organize from the regulated mobility of their dynamic protein components remains unclear. Here, we combine fluorescence spectroscopy and chemical genetics to study in living cells the diffusion of polo-like kinase 1 (PLK1), an enzyme critical for centrosome maturation at the onset of mitosis. The cytoplasmic diffusion of a functional EGFP-PLK1 fusion correlates inversely with known changes in its enzymatic activity during the cell cycle. Specific EGFP-PLK1 inhibition using chemical genetics enhances mobility, as do point mutations inactivating the polo-box or kinase domains responsible for substrate recognition and catalysis. Spatial mapping of EGFP-PLK1 diffusion across living cells, using raster image correlation spectroscopy and line scanning, detects regions of low mobility in centrosomes. These regions exhibit characteristics of increased transient recursive EGFP-PLK1 binding, distinct from the diffusion of stable EGFP-PLK1-containing complexes in the cytoplasm. Chemical genetic suppression of mitotic EGFP-PLK1 activity, even after centrosome maturation, causes defects in centrosome structure, which recover when activity is restored. Our findings imply that continuous PLK1 activity during mitosis maintains centrosome self-organization by a mechanism dependent on its reaction and diffusion, suggesting a model for the formation of stable mitotic structures using dynamic protein kinases.

Dictyostelium amoebae and neutrophils can swim.

Animal cells migrating over a substratum crawl in amoeboid fashion; how the force against the substratum is achieved remains uncertain. We find that amoebae and neutrophils, cells traditionally used to study cell migration on a solid surface, move toward a chemotactic source while suspended in solution. They can swim and do so with speeds similar to those on a solid substrate. Based on the surprisingly rapidly changing shape of amoebae as they swim and earlier theoretical schemes for how suspended microorganisms can migrate (Purcell EM (1977) Life at low Reynolds number. Am J Phys 45:3-11), we suggest the general features these cells use to gain traction with the medium. This motion requires either the movement of the cell's surface from the cell's front toward its rear or protrusions that move down the length of the elongated cell. Our results indicate that a solid substratum is not a prerequisite for these cells to produce a forward thrust during movement and suggest that crawling and swimming are similar processes, a comparison we think is helpful in understanding how cells migrate.

Multisystem inflammatory syndrome in children is driven by zonulin-dependent loss of gut mucosal barrier.

BACKGROUNDWeeks after SARS-CoV-2 infection or exposure, some children develop a severe, life-threatening illness called multisystem inflammatory syndrome in children (MIS-C). Gastrointestinal (GI) symptoms are common in patients with MIS-C, and a severe hyperinflammatory response ensues with potential for cardiac complications. The cause of MIS-C has not been identified to date.METHODSHere, we analyzed biospecimens from 100 children: 19 with MIS-C, 26 with acute COVID-19, and 55 controls. Stools were assessed for SARS-CoV-2 by reverse transcription PCR (RT-PCR), and plasma was examined for markers of breakdown of mucosal barrier integrity, including zonulin. Ultrasensitive antigen detection was used to probe for SARS-CoV-2 antigenemia in plasma, and immune responses were characterized. As a proof of concept, we treated a patient with MIS-C with larazotide, a zonulin antagonist, and monitored the effect on antigenemia and the patient's clinical response.RESULTSWe showed that in children with MIS-C, a prolonged presence of SARS-CoV-2 in the GI tract led to the release of zonulin, a biomarker of intestinal permeability, with subsequent trafficking of SARS-CoV-2 antigens into the bloodstream, leading to hyperinflammation. The patient with MIS-C treated with larazotide had a coinciding decrease in plasma SARS-CoV-2 spike antigen levels and inflammatory markers and a resultant clinical improvement above that achieved with currently available treatments.CONCLUSIONThese mechanistic data on MIS-C pathogenesis provide insight into targets for diagnosing, treating, and preventing MIS-C, which are urgently needed for this increasingly common severe COVID-19-related disease in children.

Multimodal CARS microscopy determination of the impact of diet on macrophage infiltration and lipid accumulation on plaque formation in ApoE-deficient mice.

We characterized several cellular and structural features of early stage Type II/III atherosclerotic plaques in an established model of atherosclerosis-the ApoE-deficient mouse-by using a multimodal, coregistered imaging system that integrates three nonlinear optical microscopy (NLOM) contrast mechanisms: coherent anti-Stokes Raman scattering (CARS), second harmonic generation (SHG), and two-photon excitation fluorescence (TPEF). Specifically, the infiltration of lipid-rich macrophages and the structural organization of collagen and elastin fibers were visualized by CARS, SHG, and TPEF, respectively, in thick tissue specimens without the use of exogenous labels or dyes. Label-free CARS imaging of macrophage accumulation was confirmed by histopathology using CD68 staining. A high-fat, high-cholesterol Western diet resulted in an approximate 2-fold increase in intimal plaque area, defined by CARS signals of lipid-rich macrophages. Additionally, analysis of collagen distribution within lipid-rich plaque regions revealed nearly a 4-fold decrease in the Western diet-fed mice, suggesting NLOM sensitivity to increased matrix metalloproteinase (MMP) activity and decreased smooth muscle cell (SMC) accumulation. These imaging results provide significant insight into the structure and composition of early stage Type II/III plaque during formation and allow for quantitative measurements of the impact of diet and other factors on critical plaque and arterial wall features.

Expansion microscopy of C. elegans.

We recently developed expansion microscopy (ExM), which achieves nanoscale-precise imaging of specimens at ~70 nm resolution (with ~4.5x linear expansion) by isotropic swelling of chemically processed, hydrogel-embedded tissue. ExM of C. elegans is challenged by its cuticle, which is stiff and impermeable to antibodies. Here we present a strategy, expansion of C. elegans (ExCel), to expand fixed, intact C. elegans. ExCel enables simultaneous readout of fluorescent proteins, RNA, DNA location, and anatomical structures at resolutions of ~65-75 nm (3.3-3.8x linear expansion). We also developed epitope-preserving ExCel, which enables imaging of endogenous proteins stained by antibodies, and iterative ExCel, which enables imaging of fluorescent proteins after 20x linear expansion. We demonstrate the utility of the ExCel toolbox for mapping synaptic proteins, for identifying previously unreported proteins at cell junctions, and for gene expression analysis in multiple individual neurons of the same animal.

PTH-induced internalization of apical membrane NaPi2a: role of actin and myosin VI.

Parathyroid hormone (PTH) plays a critical role in the regulation of renal phosphorous homeostasis by altering the levels of the sodium-phosphate cotransporter NaPi2a in the brush border membrane (BBM) of renal proximal tubular cells. While details of the molecular events of PTH-induced internalization of NaPi2a are emerging, the precise events governing NaPi2a removal from brush border microvilli in response to PTH remain to be fully determined. Here we use a novel application of total internal reflection fluorescence microscopy to examine how PTH induces movement of NaPi2a out of brush border microvilli in living cells in real time. We show that a dynamic actin cytoskeleton is required for NaPi2a removal from the BBM in response to PTH. In addition, we demonstrate that a myosin motor that has previously been shown to be coregulated with NaPi2a, myosin VI, is necessary for PTH-induced removal of NaPi2a from BBM microvilli.

Regulation of rat intestinal Na-dependent phosphate transporters by dietary phosphate.

Hyperphosphatemia associated with chronic kidney disease is one of the factors that can promote vascular calcification, and intestinal P(i) absorption is one of the pharmacological targets that prevents it. The type II Na-P(i) cotransporter NaPi-2b is the major transporter that mediates P(i) reabsorption in the intestine. The potential role and regulation of other Na-P(i) transporters remain unknown. We have identified expression of the type III Na-P(i) cotransporter PiT-1 in the apical membrane of enterocytes. Na-P(i) transport activity and NaPi-2b and PiT-1 proteins are mostly expressed in the duodenum and jejunum of rat small intestine; their expression is negligible in the ileum. In response to a chronic low-P(i) diet, there is an adaptive response restricted to the jejunum, with increased brush border membrane (BBM) Na-P(i) transport activity and NaPi-2b, but not PiT-1, protein and mRNA abundance. However, in rats acutely switched from a low- to a high-P(i) diet, there is an increase in BBM Na-P(i) transport activity in the duodenum that is associated with an increase in BBM NaPi-2b protein abundance. Acute adaptive upregulation is restricted to the duodenum and induces an increase in serum P(i) that produces a transient postprandial hyperphosphatemia. Our study, therefore, indicates that Na-P(i) transport activity and NaPi-2b protein expression are differentially regulated in the duodenum vs. the jejunum and that postprandial upregulation of NaPi-2b could be a potential target for treatment of hyperphosphatemia.

Differential regulation of the renal sodium-phosphate cotransporters NaPi-IIa, NaPi-IIc, and PiT-2 in dietary potassium deficiency.

Dietary potassium (K) deficiency is accompanied by phosphaturia and decreased renal brush border membrane (BBM) vesicle sodium (Na)-dependent phosphate (P(i)) transport activity. Our laboratory previously showed that K deficiency in rats leads to increased abundance in the proximal tubule BBM of the apical Na-P(i) cotransporter NaPi-IIa, but that the activity, diffusion, and clustering of NaPi-IIa could be modulated by the altered lipid composition of the K-deficient BBM (Zajicek HK, Wang H, Puttaparthi K, Halaihel N, Markovich D, Shayman J, Beliveau R, Wilson P, Rogers T, Levi M. Kidney Int 60: 694-704, 2001; Inoue M, Digman MA, Cheng M, Breusegem SY, Halaihel N, Sorribas V, Mantulin WW, Gratton E, Barry NP, Levi M. J Biol Chem 279: 49160-49171, 2004). Here we investigated the role of the renal Na-P(i) cotransporters NaPi-IIc and PiT-2 in K deficiency. Using Western blotting, immunofluorescence, and quantitative real-time PCR, we found that, in rats and in mice, K deficiency is associated with a dramatic decrease in the NaPi-IIc protein abundance in proximal tubular BBM and in NaPi-IIc mRNA. In addition, we documented the presence of a third Na-coupled P(i) transporter in the renal BBM, PiT-2, whose abundance is also decreased by dietary K deficiency in rats and in mice. Finally, electron microscopy showed subcellular redistribution of NaPi-IIc in K deficiency: in control rats, NaPi-IIc immunolabel was primarily in BBM microvilli, whereas, in K-deficient rats, NaPi-IIc BBM label was reduced, and immunolabel was prevalent in cytoplasmic vesicles. In summary, our results demonstrate that decreases in BBM abundance of the phosphate transporter NaPi-IIc and also PiT-2 might contribute to the phosphaturia of dietary K deficiency, and that the three renal BBM phosphate transporters characterized so far can be differentially regulated by dietary perturbations.

Altered renal tubular expression of the complement inhibitor Crry permits complement activation after ischemia/reperfusion.

Ischemia/reperfusion (I/R) of several organs results in complement activation, but the kidney is unique in that activation after I/R occurs only via the alternative pathway. We hypothesized that selective activation of this pathway after renal I/R could occur either because of a loss of complement inhibition or from increased local synthesis of complement factors. We examined the relationship between renal complement activation after I/R and the levels and localization of intrinsic membrane complement inhibitors. We found that loss of polarity of complement receptor 1-related protein y (Crry) in the tubular epithelium preceded activation of the alternative pathway along the basolateral aspect of the tubular cells. Heterozygous gene-targeted mice that expressed lower amounts of Crry were more sensitive to ischemic injury. Furthermore, inhibition of Crry expressed by proximal tubular epithelial cells in vitro resulted in alternative pathway-mediated injury to the cells. Thus, altered expression of a complement inhibitor within the tubular epithelium appears to be a critical factor permitting activation of the alternative pathway of complement after I/R. Increased C3 mRNA and decreased factor H mRNA were also detected in the outer medulla after I/R, suggesting that altered synthesis of these factors might further contribute to complement activation in this location.

Fluorescence correlation spectroscopy and fluorescence lifetime imaging microscopy.

With few and commercially available add-ons, both confocal and full-field fluorescence microscopes can be adapted to provide more information on the biological sample of interest. In this review we discuss the possibilities offered by two additional functionalities to fluorescence microscopes, fluorescence correlation spectroscopy (FCS) and fluorescence lifetime imaging mi croscopy (FLIM). FCS measurements at a single point in a sample allow kinetic and diffusion properties of fluorescently labeled molecules to be determined, as well as their concentration and aggregation state. Data from multiple points of the sample can be acquired using scanning-FCS, image correlation spectroscopy, and raster image correlation spectroscopy. These techniques cover phenomena with characteristic durations from sub-microsecond to second time scales. The power of FLIM lies in the fact that the measured fluorescent lifetime of a fluorophore is sensitive to the molecular environment of that fluorophore. FLIM is a robust means to quantify Forster resonance energy transfer and thus determine protein-protein interactions or protein conformational changes. In addition, FLIM is very valuable for functional imaging of ion concentrations in cells and tissues as it can be applied in heterogeneously labeled samples. In summary, FCS and FLIM allow information to be gathered beyond localization, including diffusional mobility, protein clustering and interactions, and molecular environment.

Renal phosphate-wasting disorders.

The renal regulation of phosphate is a complex process. Clinical disorders of phosphate handling have led to the identification of several genes and proteins involved in the maintenance of phosphate homeostasis. Further work is required to elucidate the precise pathways that regulate renal phosphate transport.

Preventing proteostasis diseases by selective inhibition of a phosphatase regulatory subunit.

Protein phosphorylation regulates virtually all biological processes. Although protein kinases are popular drug targets, targeting protein phosphatases remains a challenge. Here, we describe Sephin1 (selective inhibitor of a holophosphatase), a small molecule that safely and selectively inhibited a regulatory subunit of protein phosphatase 1 in vivo. Sephin1 selectively bound and inhibited the stress-induced PPP1R15A, but not the related and constitutive PPP1R15B, to prolong the benefit of an adaptive phospho-signaling pathway, protecting cells from otherwise lethal protein misfolding stress. In vivo, Sephin1 safely prevented the motor, morphological, and molecular defects of two otherwise unrelated protein-misfolding diseases in mice, Charcot-Marie-Tooth 1B, and amyotrophic lateral sclerosis. Thus, regulatory subunits of phosphatases are drug targets, a property exploited here to safely prevent two protein misfolding diseases.

Neonatal development of the stratum corneum pH gradient: localization and mechanisms leading to emergence of optimal barrier function.

Although basal permeability barrier function is established at birth, the higher risk for infections, dermatitis, and percutaneous absorption of toxic agents may indicate incomplete permeability barrier maturation in the early neonatal period. Since stratum corneum (SC) acidification in adults is required for normal permeability barrier homeostasis, and lipid processing occurs via acidic pH dependent enzymes, we hypothesized that, in parallel with the less acidic surface pH, newborn SC would exhibit signs of incomplete barrier formation. Fluorescence lifetime imaging reveals that neonatal rat SC acidification first becomes evident by postnatal day 3, in extracellular "microdomains" at the SC- stratum granulosum (SG) interface, where pH-sensitive lipid processing is known to occur. This localized acidification correlated temporally with efficient processing of secreted lamellar body contents to mature extracellular lamellar bilayers. Since expression of the key acidifying mechanism NHE1 is maximal just prior to birth, and gradually declines over the first postnatal week, suboptimal SC acidification at birth cannot be attributed to insufficient NHE1 expression, but could instead reflect reduced NHE1 activity. Expression of the key lipid processing enzyme, beta-glucocerebrosidase (beta-GlcCer'ase), develops similar to NHE1, excluding a lack of beta-GlcCer'ase protein as rate limiting for efficient lipid processing. These results define a postnatal development consisting of initial acidification in the lower SC followed by outward progression, which is accompanied by formation of mature extracellular lamellar membranes. Thus, full barrier competence appears to require the extension of acidification in microdomains from the SC/SG interface outward toward the skin surface in the immediate postnatal period.

Fluorescence lifetime imaging for the two-photon microscope: time-domain and frequency-domain methods.

Fluorescence lifetime images are obtained with the laser scanning microscope using two methods: the time-correlated single-photon counting method and the frequency-domain method. In the same microscope system, we implement both methods. We perform a comparison of the performance of the two approaches in terms of signal-to-noise ratio (SNR) and the speed of data acquisition. While in our practical implementation the time-correlated single-photon counting technique provides a better SNR for low-intensity images, the frequency-domain method is faster and provides less distortion for bright samples.

Mitigating thermal mechanical damage potential during two-photon dermal imaging.

Two-photon excitation fluorescence microscopy allows in vivo high-resolution imaging of human skin structure and biochemistry with a penetration depth over 100 microm. The major damage mechanism during two-photon skin imaging is associated with the formation of cavitation at the epidermal-dermal junction, which results in thermal mechanical damage of the tissue. In this report, we verify that this damage mechanism is of thermal origin and is associated with one-photon absorption of infrared excitation light by melanin granules present in the epidermal-dermal junction. The thermal mechanical damage threshold for selected Caucasian skin specimens from a skin bank as a function of laser pulse energy and repetition rate has been determined. The experimentally established thermal mechanical damage threshold is consistent with a simple heat diffusion model for skin under femtosecond pulse laser illumination. Minimizing thermal mechanical damage is vital for the potential use of two-photon imaging in noninvasive optical biopsy of human skin in vivo. We describe a technique to mitigate specimen thermal mechanical damage based on the use of a laser pulse picker that reduces the laser repetition rate by selecting a fraction of pulses from a laser pulse train. Since the laser pulse picker decreases laser average power while maintaining laser pulse peak power, thermal mechanical damage can be minimized while two-photon fluorescence excitation efficiency is maximized.