scholarly journals Mechanistic Determinants of Slow Axonal Transport and Presynaptic Targeting of Clathrin Packets

2020 ◽  
Author(s):  
Archan Ganguly ◽  
Florian Wernert ◽  
Sébastien Phan ◽  
Daniela Boassa ◽  
Utpal Das ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Synaptic Vesicle ◽  
Axonal Transport ◽  
Imaging Mass Spectrometry ◽  
Super Resolution ◽  
Slow Axonal Transport ◽  
Em Tomography ◽  
Overall Kinetics ◽  
Radial Organization ◽  
Membrane Infoldings

SUMMARYClathrin has established roles in endocytosis, with clathrin-cages enclosing membrane infoldings, followed by rapid disassembly and reuse of monomers. However, in neurons, clathrin synthesized in cell-bodies is conveyed into axons and synapses via slow axonal transport; as shown by classic pulse-chase radiolabeling. What is the cargo-structure, and mechanisms underlying transport and presynaptic-targeting of clathrin? What is the precise organization at synapses? Combining live-imaging, mass-spectrometry (MS), Apex-labeled EM-tomography and super-resolution, we found that unlike dendrites where clathrin transiently assembles/disassembles as expected, axons contain stable ‘transport-packets’ that move intermittently with an anterograde bias; with actin/myosin-VI as putative tethers. Transport-packets are unrelated to endocytosis, and the overall kinetics generate a slow biased flow of axonal clathrin. Synapses have integer-numbers of clathrin-packets circumferentially abutting the synaptic-vesicle cluster, advocating a model where delivery of clathrin-packets by slow axonal transport generates a radial organization of clathrin at synapses. Our experiments reveal novel trafficking mechanisms, and an unexpected nanoscale organization of synaptic clathrin.

scholarly journals Processive movement of Actin by Biased Polymerization: A new paradigm of Axonal Transport

2017 ◽  
Author(s):  
Nilaj Chakrabarty ◽  
Pankaj Dubey ◽  
Yong Tang ◽  
Archan Ganguly ◽  
Kelsey Ladt ◽  
...  
Keyword(s):  
Axonal Transport ◽  
Super Resolution ◽  
Quantitative Model ◽  
Actin Dynamics ◽  
Imaging Data ◽  
New Paradigm ◽  
Slow Axonal Transport ◽  
Local Assembly ◽  
Mechanistic Basis ◽  
Slow Transport

ABSTRACTClassic pulse-chase studies have shown that actin is conveyed in slow axonal transport, but the mechanistic basis for this movement is unknown. Recently, we reported that axonal actin was surprisingly dynamic, with focal assembly/dis-assembly events (“hotspots”) and elongating polymers along the axon-shaft (“trails”). Using a combination of live imaging, super-resolution microscopy, and modeling, here we explore how these axonal actin dynamics can lead to processive transport. We found abundant actin nucleation, along with a slow, anterogradely-biased flow of actin in axon-shafts. Starting with first principles of monomer/filament assembly – and incorporating imaging data – we generated a quantitative model simulating axonal hotspots and trails. Our simulations predict that the axonal actin dynamics indeed lead to an anterogradely-biased flow of the population, at rates consistent with slow transport. Collectively, the data point to a surprising scenario where local assembly and biased polymerization generate the slow axonal transport of actin. This mechanism is distinct from polymer-sliding, and seems well suited to convey highly dynamic cytoskeletal cargoes.AcknowledgementsThis work was supported by an NIH grant to SR (R01NS075233). The authors thank Stephanie Gupton (UNC) for the Mena/Vasp constructs.

Spatial Metabolomics of the Human Kidney Using MALDI Trapped Ion Mobility Imaging Mass Spectrometry

2020 ◽  
Author(s):  
Elizabeth Neumann ◽  
Lukasz Migas ◽  
Jamie L. Allen ◽  
Richard Caprioli ◽  
Raf Van de Plas ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Ion Mobility ◽  
Imaging Mass Spectrometry ◽  
Structural Complexity ◽  
Human Kidney ◽  
Resolving Power ◽  
Mobility Separation ◽  
Trapped Ion ◽  
Mobility Data ◽  
Ion Mobility Separation

<div> <div> <p>Small metabolites are essential for normal and diseased biological function but are difficult to study because of their inherent structural complexity. MALDI imaging mass spectrometry (IMS) of small metabolites is particularly challenging as MALDI matrix clusters are often isobaric with metabolite ions, requiring high resolving power instrumentation or derivatization to circumvent this issue. An alternative to this is to perform ion mobility separation before ion detection, enabling the visualization of metabolites without the interference of matrix ions. Here, we use MALDI timsTOF IMS to image small metabolites at high spatial resolution within the human kidney. Through this, we have found metabolites, such as arginic acid, acetylcarnitine, and choline that localize to the cortex, medulla, and renal pelvis, respectively. We have also demonstrated that trapped ion mobility spectrometry (TIMS) can resolve matrix peaks from metabolite signal and separate both isobaric and isomeric metabolites with different localizations within the kidney. The added ion mobility data dimension dramatically increased the peak capacity for molecular imaging experiments. Future work will involve further exploring the small metabolite profiles of human kidneys as a function of age, gender, and ethnicity.</p></div></div>

scholarly journals Neurodegenerative Proteinopathies in the Proteoform Spectrum—Tools and Challenges

2021 ◽  
Vol 22 (3) ◽  
pp. 1085
Author(s):  
Aneeqa Noor ◽  
Saima Zafar ◽  
Inga Zerr
Keyword(s):  
Mass Spectrometry ◽  
Neurodegenerative Disorders ◽  
Gel Electrophoresis ◽  
Large Fraction ◽  
Imaging Mass Spectrometry ◽  
Living Tissue ◽  
Disease Prognosis ◽  
Highly Sensitive ◽  
Jakob Disease ◽  
Hydrophobic Nature

Proteinopathy refers to a group of disorders defined by depositions of amyloids within living tissue. Neurodegenerative proteinopathies, including Alzheimer’s disease, Parkinson’s disease, Creutzfeldt–Jakob disease, and others, constitute a large fraction of these disorders. Amyloids are highly insoluble, ordered, stable, beta-sheet rich proteins. The emerging theory about the pathophysiology of neurodegenerative proteinopathies suggests that the primary amyloid-forming proteins, also known as the prion-like proteins, may exist as multiple proteoforms that contribute differentially towards the disease prognosis. It is therefore necessary to resolve these disorders on the level of proteoforms rather than the proteome. The transient and hydrophobic nature of amyloid-forming proteins and the minor post-translational alterations that lead to the formation of proteoforms require the use of highly sensitive and specialized techniques. Several conventional techniques, like gel electrophoresis and conventional mass spectrometry, have been modified to accommodate the proteoform theory and prion-like proteins. Several new ones, like imaging mass spectrometry, have also emerged. This review aims to discuss the proteoform theory of neurodegenerative disorders along with the utility of these proteomic techniques for the study of highly insoluble proteins and their associated proteoforms.

scholarly journals Imaging Mass Spectrometry-Based Proteomic Analysis to Differentiate Melanocytic Nevi and Malignant Melanoma

Cancers ◽  
2021 ◽  
Vol 13 (13) ◽  
pp. 3197
Author(s):  
Rita Casadonte ◽  
Mark Kriegsmann ◽  
Katharina Kriegsmann ◽  
Isabella Hauk ◽  
Rolf R. Meliß ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Malignant Melanoma ◽  
Operating Characteristic ◽  
Characteristic Curve ◽  
Imaging Mass Spectrometry ◽  
Independent Set ◽  
Molecular Techniques ◽  
Charge Ratio ◽  
Linear Discriminant ◽  
Melanocytic Nevi

The discrimination of malignant melanoma from benign nevi may be difficult in some cases. For this reason, immunohistological and molecular techniques are included in the differential diagnostic toolbox for these lesions. These methods are time consuming when applied subsequently and, in some cases, no definitive diagnosis can be made. We studied both lesions by imaging mass spectrometry (IMS) in a large cohort (n = 203) to determine a different proteomic profile between cutaneous melanomas and melanocytic nevi. Sample preparation and instrument setting were tested to obtain optimal results in term of data quality and reproducibility. A proteomic signature was found by linear discriminant analysis to discern malignant melanoma from benign nevus (n = 113) with an overall accuracy of >98%. The prediction model was tested in an independent set (n = 90) reaching an overall accuracy of 93% in classifying melanoma from nevi. Statistical analysis of the IMS data revealed mass-to-charge ratio (m/z) peaks which varied significantly (Area under the receiver operating characteristic curve > 0.7) between the two tissue types. To our knowledge, this is the largest IMS study of cutaneous melanoma and nevi performed up to now. Our findings clearly show that discrimination of melanocytic nevi from melanoma is possible by IMS.

scholarly journals Kinesin Mutations Cause Motor Neuron Disease Phenotypes by Disrupting Fast Axonal Transport in Drosophila

Genetics ◽  
1996 ◽  
Vol 144 (3) ◽  
pp. 1075-1085 ◽  
Author(s):  
Daryl D Hurd ◽  
William M Saxton
Keyword(s):  
Motor Neuron ◽  
Axonal Transport ◽  
Action Potentials ◽  
Light And Electron Microscopy ◽  
Synaptic Membrane ◽  
Slow Axonal Transport ◽  
Fast Axonal Transport ◽  
Motor Neuron Diseases ◽  
Transmitter Secretion ◽  
Axon Terminals

Abstract Previous work has shown that mutation of the gene that encodes the microtubule motor subunit kinesin heavy chain (Khc) in Drosophila inhibits neuronal sodium channel activity, action potentials and neurotransmitter secretion. These physiological defects cause progressive distal paralysis in larvae. To identify the cellular defects that cause these phenotypes, larval nerves were studied by light and electron microscopy. The axons of Khc mutants develop dramatic focal swellings along their lengths. The swellings are packed with fast axonal transport cargoes including vesicles, synaptic membrane proteins, mitochondria and prelysosomal organelles, but not with slow axonal transport cargoes such as cytoskeletal elements. Khc mutations also impair the development of larval motor axon terminals, causing dystrophic morphology and marked reductions in synaptic bouton numbers. These observations suggest that as the concentration of maternally provided wild-type KHC decreases, axonal organelles transported by kinesin periodically stall. This causes organelle jams that disrupt retrograde as well as anterograde fast axonal transport, leading to defective action potentials, dystrophic terminals, reduced transmitter secretion and progressive distal paralysis. These phenotypes parallel the pathologies of some vertebrate motor neuron diseases, including some forms of amyotrophic lateral sclerosis (ALS), and suggest that impaired fast axonal transport is a key element in those diseases.

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