Super-resolution microscopy unveils transmembrane domain-mediated internalization of cross-reacting material 197 into diphtheria toxin-resistant mouse J774A.1 cells and primary rat fibroblasts in vitro

Vectors based on the formation of a soluble DNA–polycation complex are being developed for the treatment of human diseases. These complexes are rapidly taken up by receptor-mediated endocytosis, but are inefficiently delivered to the nucleus owing to entrapment in membrane-bound vesicles. In this study we introduced the transmembrane domain of diphtheria toxin into a DNA–polycation conjugate complex in an effort to increase gene transfer by membrane perturbation. The transmembrane domain of diphtheria toxin was expressed in Escherichia coli as a maltose-binding protein fusion and chemically coupled to high-molecular-mass poly-l-lysine. Incorporation of this conjugate into a traditional complex formed with a luciferase-containing plasmid with an asialo-orosomucoid–polycation conjugate significantly increased transfection efficiency in vitro in a manner proportional to the amount of diphtheria toxin incorporated. The delivery of luciferase RNA transcript was similarly increased when complexed with similar polycation conjugates. This study uses the structural biology of a bacterial protein to improve polycation-based gene delivery.

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Radial F-actin Organization During Early Neuronal Development

10.1101/372813 ◽

2018 ◽

Author(s):

Durga Praveen Meka ◽

Robin Scharrenberg ◽

Bing Zhao ◽

Theresa König ◽

Irina Schaefer ◽

...

Keyword(s):

Neuronal Development ◽

Super Resolution ◽

Actin Dynamics ◽

Cell Periphery ◽

Microtubule Organizing Center ◽

Actin Organization ◽

Organizing Center ◽

Super Resolution Microscopy ◽

Radial Organization

AbstractThe centrosome is thought to be the major neuronal microtubule-organizing center (MTOC) in early neuronal development, producing microtubules with a radial organization. In addition, albeit in vitro, recent work showed that isolated centrosomes could serve as an actin-organizing center (Farina et al., 2016), raising the possibility that neuronal development may, in addition, require a centrosome-based actin radial organization. Here we report, using super-resolution microscopy and live-cell imaging, F-actin organization around the centrosome with dynamic F-actin aster-like structures with F-actin fibers extending and retracting actively. Photoconversion/photoactivation experiments and molecular manipulations of F-actin stability reveal a robust flux of somatic F-actin towards the cell periphery. Finally, we show that somatic F-actin intermingles with centrosomal PCM-1 satellites. Knockdown of PCM-1 and disruption of centrosomal activity not only affect F-actin dynamics near the centrosome but also in distal growth cones. Collectively the data show a radial F-actin organization during early neuronal development, which might be a cellular mechanism for providing peripheral regions with a fast and continuous source of actin polymers; hence sustaining initial neuronal development.

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Mutant EGFR is a preferred SMURF2 substrate of ubiquitination: role in enhanced receptor stability and TKI sensitivity

10.1101/2020.04.02.022012 ◽

2020 ◽

Author(s):

Paramita Ray ◽

Krishnan Raghunathan ◽

Aarif Ahsan ◽

Uday Sankar Allam ◽

Shirish Shukla ◽

...

Keyword(s):

Growth Factor Receptor ◽

Super Resolution ◽

Receptor Internalization ◽

Stochastic Optical Reconstruction Microscopy ◽

Steady State Levels ◽

Epidermal Growth ◽

Optical Reconstruction ◽

Super Resolution Microscopy

ABSTRACTWe previously reported that differential protein degradation of TKI-sensitive [L858R, del(E746-A750)] and resistant (T790M) epidermal growth factor receptor (EGFR) mutants upon erlotinib treatment correlates with drug sensitivity. However, the molecular mechanism remains unclear. We also reported SMAD ubiquitination regulatory factor 2 (SMURF2) ligase activity is important in stabilizing EGFR. Here, using in vitro and in vivo ubiquitination assays, mass spectrometry, and super-resolution microscopy, we show SMURF2-EGFR functional interaction is critical in receptor stability and TKI sensitivity. We found that L858R/T790M EGFR is a preferred substrate of SMURF2-UBCH5 (an E3-E2) complex-mediated K63-linked polyubiquitination, which preferentially stabilizes mutant receptor. We identified four lysine (K) residues (K721, 846, 1037 and 1164) as the sites of ubiquitination and replacement of K to acetylation-mimicking asparagine (Q) at K1037 position in L858R/T790M background converts the stable protein sensitive to erlotinib-induced degradation. Using STochastic Optical Reconstruction Microscopy (STORM) imaging, we show that SMURF2 presence allows longer membrane retention of activated EGFR upon EGF treatment, whereas, siRNA-mediated SMURF2 knockdown fastens receptor endocytosis and lysosome enrichment. In an erlotinib-sensitive PC9 cells, SMURF2 overexpression increased EGFR levels with improved erlotinib tolerance, whereas, SMURF2 knockdown decreased EGFR steady state levels in NCI-H1975 and PC9-AR cells to overcome erlotinib and AZD-9291 resistance respectively. Additionally, by genetically altering the SMURF2-UBCH5 complex formation destabilized EGFR. Together, we propose that SMURF2-mediated preferential polyubiquitination of L858R/T790M EGFR may be competing with acetylation-mediated receptor internalization to provide enhanced receptor stability and that disruption of the E3-E2 complex may be an attractive alternate to overcome TKI resistance.

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Binding of the RNA Chaperone Hfq on Target mRNAs Promotes the Small RNA RyhB-Induced Degradation in Escherichia coli

Non-Coding RNA ◽

10.3390/ncrna7040064 ◽

2021 ◽

Vol 7 (4) ◽

pp. 64

Author(s):

David Lalaouna ◽

Karine Prévost ◽

Seongjin Park ◽

Thierry Chénard ◽

Marie-Pier Bouchard ◽

...

Keyword(s):

Complex Formation ◽

Super Resolution ◽

Rnase E ◽

Rna Chaperone ◽

Target Mrnas ◽

Mrna Targets ◽

Super Resolution Microscopy

Many RNA-RNA interactions depend on molecular chaperones to form and remain stable in living cells. A prime example is the RNA chaperone Hfq, which is a critical effector involved in regulatory interactions between small RNAs (sRNAs) and cognate target mRNAs in Enterobacteriaceae. While there is a great deal of in vitro biochemical evidence supporting the model that Hfq enhances rates or affinities of sRNA:mRNA interactions, there is little corroborating in vivo evidence. Here we used in vivo tools including reporter genes, co-purification assays, and super-resolution microscopy to analyze the role of Hfq in RyhB-mediated regulation, and we found that Hfq is often unnecessary for efficient RyhB:mRNA complex formation in vivo. Remarkably, our data suggest that a primary function of Hfq is to promote RyhB-induced cleavage of mRNA targets by RNase E. Moreover, our work indicates that Hfq plays a more limited role in dictating regulatory outcomes following sRNAs RybB and DsrA complex formation with specific target mRNAs. Our investigation helps evaluate the roles played by Hfq in some RNA-mediated regulation.

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Chromosome segregation is driven by joint microtubule sliding action of kinesins KIF4A and EG5

10.1101/863381 ◽

2019 ◽

Cited By ~ 2

Author(s):

Kruno Vukušić ◽

Renata Buđa ◽

Ivana Ponjavić ◽

Patrik Risteski ◽

Iva M. Tolić

Keyword(s):

Cell Division ◽

Molecular Mechanism ◽

Chromosome Segregation ◽

Super Resolution ◽

Human Cells ◽

Microtubule Stability ◽

Sliding Mechanism ◽

Spindle Elongation ◽

Super Resolution Microscopy

Successful cell division requires proper chromosome segregation during anaphase. Forces required for chromosome segregation in human cells are linked to sliding of antiparallel microtubules and sliding capacity has been demonstrated in vitro for multiple motor proteins, but the molecular mechanism of sliding in the spindle of human cells remains unknown. Using combined depletion and inactivation assays to explore redundancy between multiple targets together with CRISPR technology, we found that PRC1-dependent motor KIF4A/kinesin-4, together with EG5/kinesin-5 motor is essential for spindle elongation in human cells. Photoactivation of tubulin and super-resolution microscopy show that perturbation of both proteins impairs sliding, while decreased midzone microtubule stability cannot explain the observed anaphase arrest. Thus, two independent sliding modules power sliding mechanism that drives spindle elongation in human cells.

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Hyperphosphorylated tau self-assembles into amorphous aggregates eliciting TLR4-dependent inflammatory responses

10.21203/rs.3.rs-374549/v1 ◽

2021 ◽

Author(s):

David Klenerman ◽

Jonathan Meng ◽

Yu Zhang ◽

Dominik Saman ◽

Suman De ◽

...

Keyword(s):

Inflammatory Responses ◽

Mechanistic Model ◽

Super Resolution ◽

Hyperphosphorylated Tau ◽

Toll Like Receptor ◽

Wild Type ◽

Toll Like Receptor 4 ◽

Soluble Aggregates ◽

Super Resolution Microscopy

Abstract Soluble aggregates of the microtubule-associated protein tau have been challenging to assemble and characterize, despite their important role in the development of tauopathies. We found that sequential hyperphosphorylation by PKA in conjugation with either GSK3-β or SAPK4 enabled recombinant wild-type (WT) tau of isoform 0N4R to spontaneously polymerize into small amorphous aggregates in vitro. We employed tandem mass spectrometry to determine the phosphorylation sites and the degree of phosphorylation, and super-resolution microscopy and electron microscopy to characterize the morphology of aggregates formed. Functionally, in comparison with the unmodified aggregates, which require heparin induction to assemble, these self-assembled hyperphosphorylated tau aggregates more efficiently disrupt membrane bilayers and induce Toll-like receptor 4 (TLR4)-dependent inflammatory responses. Together, our results demonstrate that tau hyperphosphorylation is potentially damaging to cells, providing a mechanistic model of how hyperphosphorylation of tau aggregates drives neuroinflammation in tauopathies.

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Advances in pancreatic islet monolayer culture on glass surfaces enable super-resolution microscopy and insights into beta cell ciliogenesis and proliferation

Scientific Reports ◽

10.1038/srep45961 ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 13

Author(s):

Edward A. Phelps ◽

Chiara Cianciaruso ◽

Jaime Santo-Domingo ◽

Miriella Pasquier ◽

Gabriele Galliverti ◽

...

Keyword(s):

Beta Cells ◽

Pancreatic Islet ◽

Culture Media ◽

Islet Cells ◽

Cell Monolayer ◽

Super Resolution ◽

Cellular Processes ◽

Glass Surfaces ◽

Super Resolution Microscopy

Abstract A robust and reproducible method for culturing monolayers of adherent and well-spread primary islet cells on glass coverslips is required for detailed imaging studies by super-resolution and live-cell microscopy. Guided by an observation that dispersed islet cells spread and adhere well on glass surfaces in neuronal co-culture and form a monolayer of connected cells, we demonstrate that in the absence of neurons, well-defined surface coatings combined with components of neuronal culture media collectively support robust attachment and growth of primary human or rat islet cells as monolayers on glass surfaces. The islet cell monolayer cultures on glass stably maintain distinct mono-hormonal insulin+, glucagon+, somatostatin+ and PP+ cells and glucose-responsive synchronized calcium signaling as well as expression of the transcription factors Pdx-1 and NKX-6.1 in beta cells. This technical advance enabled detailed observation of sub-cellular processes in primary human and rat beta cells by super-resolution microscopy. The protocol is envisaged to have broad applicability to sophisticated analyses of pancreatic islet cells that reveal new biological insights, as demonstrated by the identification of an in vitro protocol that markedly increases proliferation of primary beta cells and is associated with a reduction in ciliated, ostensibly proliferation-suppressed beta cells.

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The Role of Taste Receptor mTAS1R3 in Chemical Communication of Gametes

International Journal of Molecular Sciences ◽

10.3390/ijms21072651 ◽

2020 ◽

Vol 21 (7) ◽

pp. 2651

Author(s):

Michaela Frolikova ◽

Tereza Otcenaskova ◽

Eliska Valasková ◽

Pavla Postlerova ◽

Romana Stopkova ◽

...

Keyword(s):

Chemical Communication ◽

Reproductive Tract ◽

Taste Receptor ◽

Super Resolution ◽

Female Reproductive Tract ◽

Male Gametes ◽

Mouse Taste ◽

Super Resolution Microscopy

Fertilization is a multiple step process leading to the fusion of female and male gametes and the formation of a zygote. Besides direct gamete membrane interaction via binding receptors localized on both oocyte and sperm surface, fertilization also involves gamete communication via chemical molecules triggering various signaling pathways. This work focuses on a mouse taste receptor, mTAS1R3, encoded by the Tas1r3 gene, as a potential receptor mediating chemical communication between gametes using the C57BL/6J lab mouse strain. In order to specify the role of mTAS1R3, we aimed to characterize its precise localization in testis and sperm using super resolution microscopy. The testis cryo-section, acrosome-intact sperm released from cauda epididymis and sperm which underwent the acrosome reaction (AR) were evaluated. The mTAS1R3 receptor was detected in late spermatids where the acrosome was being formed and in the acrosomal cap of acrosome intact sperm. AR is triggered in mice during sperm maturation in the female reproductive tract and by passing through the egg surroundings such as cumulus oophorus cells. This AR onset is independent of the extracellular matrix of the oocyte called zona pellucida. After AR, the relocation of mTAS1R3 to the equatorial segment was observed and the receptor remained exposed to the outer surroundings of the female reproductive tract, where its physiological ligand, the amino acid L-glutamate, naturally occurs. Therefore, we targeted the possible interaction in vitro between the mTAS1R3 and L-glutamate as a part of chemical communication between sperm and egg and used an anti-mTAS1R3-specific antibody to block it. We detected that the acrosome reacted spermatozoa showed a chemotactic response in the presence of L-glutamate during and after the AR, and it is likely that mTAS1R3 acted as its mediator.

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Serotonin-specific neurons differentiated from human iPSCs form distinct subtypes with synaptic protein assembly

Journal of Neural Transmission ◽

10.1007/s00702-021-02303-5 ◽

2021 ◽

Vol 128 (2) ◽

pp. 225-241

Author(s):

Charline Jansch ◽

Georg C. Ziegler ◽

Andrea Forero ◽

Sina Gredy ◽

Sina Wäldchen ◽

...

Keyword(s):

Tryptophan Hydroxylase ◽

Neuronal Cell ◽

Super Resolution ◽

Neuropsychiatric Disorders ◽

Human Ipscs ◽

Heterogeneous Nature ◽

Induced Pluripotent ◽

Super Resolution Microscopy ◽

The Brain

AbstractHuman induced pluripotent stem cells (hiPSCs) have revolutionized the generation of experimental disease models, but the development of protocols for the differentiation of functionally active neuronal subtypes with defined specification is still in its infancy. While dysfunction of the brain serotonin (5-HT) system has been implicated in the etiology of various neuropsychiatric disorders, investigation of functional human 5-HT specific neurons in vitro has been restricted by technical limitations. We describe an efficient generation of functionally active neurons from hiPSCs displaying 5-HT specification by modification of a previously reported protocol. Furthermore, 5-HT specific neurons were characterized using high-end fluorescence imaging including super-resolution microscopy in combination with electrophysiological techniques. Differentiated hiPSCs synthesize 5-HT, express specific markers, such as tryptophan hydroxylase 2 and 5-HT transporter, and exhibit an electrophysiological signature characteristic of serotonergic neurons, with spontaneous rhythmic activities, broad action potentials and large afterhyperpolarization potentials. 5-HT specific neurons form synapses reflected by the expression of pre- and postsynaptic proteins, such as Bassoon and Homer. The distribution pattern of Bassoon, a marker of the active zone along the soma and extensions of neurons, indicates functionality via volume transmission. Among the high percentage of 5-HT specific neurons (~ 42%), a subpopulation of CDH13 + cells presumably designates dorsal raphe neurons. hiPSC-derived 5-HT specific neuronal cell cultures reflect the heterogeneous nature of dorsal and median raphe nuclei and may facilitate examining the association of serotonergic neuron subpopulations with neuropsychiatric disorders.

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Super-Resolution Imaging of the Filtration Barrier Suggests a Role for Podocin R229Q in Genetic Predisposition to Glomerular Disease

Journal of the American Society of Nephrology ◽

10.1681/asn.2020060858 ◽

2021 ◽

pp. ASN.2020060858

Author(s):

Linus Butt ◽

David Unnersjö-Jess ◽

Martin Höhne ◽

Robert Hahnfeldt ◽

Dervla Reilly ◽

...

Keyword(s):

Genetic Predisposition ◽

Complex Disease ◽

Genetic Diagnosis ◽

Super Resolution ◽

Genetic Alterations ◽

Adult Patients ◽

Glomerular Injury ◽

Filtration Barrier ◽

Super Resolution Microscopy

Background Diseases of the kidney's glomerular filtration barrier are a leading cause of end-stage renal failure. Despite of a growing understanding of genes involved in glomerular disorders in children, the vast majority of adult patients lack a clear genetic diagnosis. The protein podocin p.R229Q, which results from the most common missense variant in NPHS2, is enriched in focal segmental glomerulosclerosis (FSGS) patient cohorts. However, p.R229Q has been proposed to cause disease only when trans-associated to specific additional genetic alterations, and population-based epidemiologic studies on its association with albuminuria yielded ambiguous results. Methods To test whether podocin p.R229Q may also predispose to the complex disease pathogenesis in adults, we introduced the exact genetic alteration in mice using CRISPR/Cas9-based genome editing (PodR231Q). We assessed the phenotype using super-resolution microscopy and albuminuria measurements, and evaluated the stability of the mutant protein in cell culture experiments. Results Heterozygous PodR231Q/wildtype mice did not present any overt kidney disease or proteinuria. However, homozygous PodR231Q/R231Q mice developed increased levels of albuminuria with age, and super-resolution microscopy revealed preceding ultrastructural morphologic alterations that were recently linked to disease predisposition. When injected with nephrotoxic serum to induce glomerular injury, heterozygous PodR231Q/wildtype mice showed a more severe course of disease compared with Podwildtype/wildtype mice. Podocin protein levels were decreased in PodR231Q/wildtype and PodR231Q/R231Q mice as well as in human cultured podocytes expressing the podocinR231Q variant. Our in vitro experiments indicate an underlying increased proteasomal degradation Conclusions Our findings demonstrate that podocin R231Q exerts a pathogenic effect on its own, supporting the concept of podocin R229Q contributing to genetic predisposition in adult patients

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