scholarly journals Size-dependent secondary nucleation and amplification of α-synuclein amyloid fibrils

2021 ◽  
Author(s):  
Arunima Sakunthala ◽  
Debalina Datta ◽  
Ambuja Navalkar ◽  
Laxmikant Gadhe ◽  
Pradeep Kadu ◽  
...  
Keyword(s):  
Amyloid Fibrils ◽  
Super Resolution ◽  
Cellular Internalization ◽  
Biological Processes ◽  
Secondary Nucleation ◽  
Size Dependent ◽  
Distinct Mechanism ◽  
In Vitro Reconstitution ◽  
Super Resolution Microscopy

The size of the amyloid seeds is known to modulate their autocatalytic amplification and cellular toxicity. However, the seed size-dependent secondary nucleation mechanism, toxicity, and disease-associated biological processes mediated by α-synuclein (α-Syn) fibrils are largely unknown. Using the cellular model and in vitro reconstitution, we showed that the size of α-Syn fibril seeds not only dictates its cellular internalization and associated cell death; but also the distinct mechanisms of fibril amplification pathways involved in the pathological conformational change of α-Syn. Specifically, small-sized fibril seeds showed elongation possibly through monomer addition at the fibril termini; whereas longer fibrils template the fibril amplification by surface-mediated nucleation as demonstrated by super-resolution microscopy. The distinct mechanism of fibril amplification, and cellular uptake along with toxicity suggest that breakage of fibrils into different sizes of seeds determine the underlying pathological outcome of synucleinopathies.

scholarly journals Affimer proteins are versatile and renewable affinity reagents

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Christian Tiede ◽  
Robert Bedford ◽  
Sophie J Heseltine ◽  
Gina Smith ◽  
Imeshi Wijetunga ◽  
...  
Keyword(s):  
Protein Function ◽  
Cell Biology ◽  
Super Resolution ◽  
Biological Processes ◽  
Affinity Reagents ◽  
Tumour Antigens ◽  
Target Molecules ◽  
Super Resolution Microscopy

Molecular recognition reagents are key tools for understanding biological processes and are used universally by scientists to study protein expression, localisation and interactions. Antibodies remain the most widely used of such reagents and many show excellent performance, although some are poorly characterised or have stability or batch variability issues, supporting the use of alternative binding proteins as complementary reagents for many applications. Here we report on the use of Affimer proteins as research reagents. We selected 12 diverse molecular targets for Affimer selection to exemplify their use in common molecular and cellular applications including the (a) selection against various target molecules; (b) modulation of protein function in vitro and in vivo; (c) labelling of tumour antigens in mouse models; and (d) use in affinity fluorescence and super-resolution microscopy. This work shows that Affimer proteins, as is the case for other alternative binding scaffolds, represent complementary affinity reagents to antibodies for various molecular and cell biology applications.

scholarly journals Radial F-actin Organization During Early Neuronal Development

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.

scholarly journals Secondary nucleation of monomers on fibril surface dominatesα-synuclein aggregation and provides autocatalytic amyloid amplification

2017 ◽  
Vol 50 ◽  
Author(s):  
Ricardo Gaspar ◽  
Georg Meisl ◽  
Alexander K. Buell ◽  
Laurence Young ◽  
Clemens F. Kaminski ◽  
...  
Keyword(s):  
Significant Contribution ◽  
Lewy Bodies ◽  
Monomer Concentration ◽  
Super Resolution ◽  
Secondary Process ◽  
Secondary Nucleation ◽  
Lewy Neurites ◽  
Rapid Generation ◽  
Differential Sedimentation ◽  
Super Resolution Microscopy

AbstractParkinson's disease (PD) is characterized by proteinaceous aggregates named Lewy Bodies and Lewy Neurites containingα-synuclein fibrils. The underlying aggregation mechanism of this protein is dominated by a secondary process at mildly acidic pH, as in endosomes and other organelles. This effect manifests as a strong acceleration of the aggregation in the presence of seeds and a weak dependence of the aggregation rate on monomer concentration. The molecular mechanism underlying this process could be nucleation of monomers on fibril surfaces or fibril fragmentation. Here, we aim to distinguish between these mechanisms. The nature of the secondary processes was investigated using differential sedimentation analysis, trap and seed experiments, quartz crystal microbalance experiments and super-resolution microscopy. The results identify secondary nucleation of monomers on the fibril surface as the dominant secondary process leading to rapid generation of new aggregates, while no significant contribution from fragmentation was found. The newly generated oligomeric species quickly elongate to further serve as templates for secondary nucleation and this may have important implications in the spreading of PD.

scholarly journals Heparin acts as a structural component of β-endorphin amyloid fibrils rather than a simple aggregation promoter

2017 ◽  
Vol 53 (7) ◽  
pp. 1273-1276 ◽  
Author(s):  
N. Nespovitaya ◽  
P. Mahou ◽  
R. F. Laine ◽  
G. S. Kaminski Schierle ◽  
C. F. Kaminski
Keyword(s):  
Structural Component ◽  
Amyloid Fibrils ◽  
Molecular Level ◽  
Super Resolution ◽  
Super Resolution Microscopy

Super-resolution microscopy gives molecular level insights into the heparin-promoted aggregation of β-endorphin amyloid fibrils.

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

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.

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

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.

scholarly journals Secondary Nucleation and the Conservation of Structural Characteristics of Amyloid Fibril Strains

2021 ◽  
Vol 8 ◽  
Author(s):  
Saeid Hadi Alijanvand ◽  
Alessia Peduzzo ◽  
Alexander K. Buell
Keyword(s):  
Amyloid Fibrils ◽  
Amyloid Fibril ◽  
Structural Characteristics ◽  
Secondary Nucleation ◽  
Primary Nucleation ◽  
Structural Preferences ◽  
Initial Seed ◽  
The Brain

Amyloid fibrils are ordered protein aggregates and a hallmark of many severe neurodegenerative diseases. Amyloid fibrils form through primary nucleation from monomeric protein, grow through monomer addition and proliferate through fragmentation or through the nucleation of new fibrils on the surface of existing fibrils (secondary nucleation). It is currently still unclear how amyloid fibrils initially form in the brain of affected individuals and how they are amplified. A given amyloid protein can sometimes form fibrils of different structure under different solution conditions in vitro, but often fibrils found in patients are highly homogeneous. These findings suggest that the processes that amplify amyloid fibrils in vivo can in some cases preserve the structural characteristics of the initial seed fibrils. It has been known for many years that fibril growth by monomer addition maintains the structure of the seed fibril, as the latter acts as a template that imposes its fold on the newly added monomer. However, for fibrils that are formed through secondary nucleation it was, until recently, not clear whether the structure of the seed fibril is preserved. Here we review the experimental evidence on this question that has emerged over the last years. The overall picture is that the fibril strain that forms through secondary nucleation is mostly defined by the solution conditions and intrinsic structural preferences, and not by the seed fibril strain.

scholarly journals Subcellular drug targeting illuminates local kinase action

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Paula J Bucko ◽  
Chloe K Lombard ◽  
Lindsay Rathbun ◽  
Irvin Garcia ◽  
Akansha Bhat ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Super Resolution ◽  
Biological Processes ◽  
Anchoring Proteins ◽  
Intracellular Compartments ◽  
Mitotic Spindle Assembly ◽  
Targeted Inhibition ◽  
Super Resolution Microscopy

Deciphering how signaling enzymes operate within discrete microenvironments is fundamental to understanding biological processes. A-kinase anchoring proteins (AKAPs) restrict the range of action of protein kinases within intracellular compartments. We exploited the AKAP targeting concept to create genetically encoded platforms that restrain kinase inhibitor drugs at distinct subcellular locations. Local Kinase Inhibition (LoKI) allows us to ascribe organelle-specific functions to broad specificity kinases. Using chemical genetics, super resolution microscopy, and live-cell imaging we discover that centrosomal delivery of Polo-like kinase 1 (Plk1) and Aurora A (AurA) inhibitors attenuates kinase activity, produces spindle defects, and prolongs mitosis. Targeted inhibition of Plk1 in zebrafish embryos illustrates how centrosomal Plk1 underlies mitotic spindle assembly. Inhibition of kinetochore-associated pools of AurA blocks phosphorylation of microtubule-kinetochore components. This versatile precision pharmacology tool enhances investigation of local kinase biology.

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

2019 ◽  
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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