<scp>KymoAnalyzer</scp>: a software tool for the quantitative analysis of intracellular transport in neurons

Neumann, Sylvia; Chassefeyre, Romain; Campbell, George E.; Encalada, Sandra E.

doi:10.1111/tra.12456

Cited by 88 publications

(78 citation statements)

References 43 publications

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“…Three hours after the stimulation, live cell imaging was performed using 40ϫ objective lens on a Nikon A1r confocal microscope. KymoAnalyzer (Neumann et al, 2017) was performed to generate kymographs and determine the velocity of the axonal transport.…”

Section: Methodsmentioning

confidence: 99%

Limiting Neuronal Nogo Receptor 1 Signaling during Experimental Autoimmune Encephalomyelitis Preserves Axonal Transport and Abrogates Inflammatory Demyelination

Lee

Kim²,

Thomas³

et al. 2019

J. Neurosci.

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We previously identified that ngr1 allele deletion limits the severity of experimental autoimmune encephalomyelitis (EAE) by preserving axonal integrity. However, whether this favorable outcome observed in EAE is a consequence of an abrogated neuronal-specific pathophysiological mechanism, is yet to be defined. Here we show that, Cre-loxP-mediated neuron-specific deletion of ngr1 preserved axonal integrity, whereas its re-expression in ngr1 ؊/؊ female mice potentiated EAE-axonopathy. As a corollary, myelin integrity was preserved under Cre deletion in ngr1 flx/flx , retinal ganglion cell axons whereas, significant demyelination occurred in the ngr1 ؊/؊ optic nerves following the re-introduction of NgR1. Moreover, Cre-loxP-mediated axon-specific deletion of ngr1 in ngr1 flx/flx mice also demonstrated efficient anterograde transport of fluorescently-labeled ChTx␤ in the optic nerves of EAE-induced mice. However, the anterograde transport of ChTx␤ displayed accumulation in optic nerve degenerative axons of EAE-induced ngr1 ؊/؊ mice, when NgR1 was reintroduced but was shown to be transported efficiently in the contralateral non-recombinant adeno-associated virus serotype 2-transduced optic nerves of these mutant mice. We further identified that the interaction between the axonal motor protein, Kinesin-1 and collapsin response mediator protein 2 (CRMP2) was unchanged upon Cre deletion of ngr1. Whereas, this Kinesin-1/CRMP2 association was reduced when NgR1 was re-expressed in the ngr1 ؊/؊ optic nerves. Our data suggest that NgR1 governs axonal degeneration in the context of inflammatory-mediated demyelination through the phosphorylation of CRMP2 by stalling axonal vesicular transport. Moreover, axon-specific deletion of ngr1 preserves axonal transport mechanisms, blunting the induction of inflammatory demyelination and limiting the severity of EAE. Key words: axonal degeneration; collapsin response mediator protein 2; demyelination; experimental autoimmune encephalomyelitis; Kinesin-1; Nogo receptor 1 Significance StatementMultiple sclerosis (MS) is commonly induced by aberrant immune-mediated destruction of the protective sheath of nerve fibers (known as myelin). However, it has been shown that MS lesions do not only consist of this disease pattern, exhibiting heterogeneity with continual destruction of axons. Here we investigate how neuronal NgR1 can drive inflammatory-mediated axonal degeneration and demyelination within the optic nerve by analyzing its downstream signaling events that govern axonal vesicular transport. We identify that abrogating the NgR1/pCRMP2 signaling cascade can maintain Kinesin-1-dependent anterograde axonal transport to limit inflammatory-mediated axonopathy and demyelination. The ability to differentiate between primary and secondary mechanisms of axonal degeneration may uncover therapeutic strategies to limit axonal damage and progressive MS.

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Section: Methodsmentioning

confidence: 99%

Limiting Neuronal Nogo Receptor 1 Signaling during Experimental Autoimmune Encephalomyelitis Preserves Axonal Transport and Abrogates Inflammatory Demyelination

Lee

Kim²,

Thomas³

et al. 2019

J. Neurosci.

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“…Run lengths and durations were analyzed by ImageJ (MTrackJ) using a pixel size of 71.0 nm. Kymographs were generated using Kymo-analyzer package (21). To ensure that run lengths were reliably captured, only run lengths greater than 200 nm were analyzed, and to estimate the average run length, this minimum distance was subtracted from all runs.…”

Section: Single Molecule Experimentsmentioning

confidence: 99%

Motor reattachment kinetics play a dominant role in multimotor-driven cargo transport

Feng

Mickolajczyk

Chen

et al. 2017

Preprint

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Kinesin-based cargo transport in cells frequently involves the coordinated activity of multiple motors, including kinesins from different families that move at different speeds. However, compared to the progress at the single-molecule level, mechanisms by which multiple kinesins coordinate their activity during cargo transport are poorly understood. To understand these multi-motor coordination mechanisms, defined pairs of kinesin-1 and kinesin-2 motors were assembled on DNA scaffolds and their motility examined in vitro. Although less processive than kinesin-1 at the single-molecule level, addition of kinesin-2 motors more effectively amplified cargo run lengths. By applying the law of total expectation to cargo binding durations in ADP, the kinesin-2 microtubule reattachment rate was shown to be 4-fold faster than that of kinesin-1. This difference in microtubule binding rates was also observed in solution by stopped-flow. High-resolution tracking of gold-nanoparticle-labeled cargo with 1 ms and 2 nm precision revealed that kinesin-2 motors detach and rebind to the microtubule much more frequently than do kinesin-1. Finally, cargo transported by kinesin-2 motors more effectively navigated roadblocks on the microtubule track. These results highlight the importance of motor reattachment kinetics during multi-motor transport and suggest a coordinated transport model in which kinesin-1 motors step effectively against loads while kinesin-2 motors rapidly unbind and rebind to the microtubule. This dynamic tethering by kinesin-2 maintains the cargo near the microtubule and enables effective navigation along crowded microtubules.

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“…Such a process is depicted in Fig. 1 and is inspired by data from kymograph readouts of cargo movement as seen in Encalada et al 2011and analyzed by a tool developed by Neumann et al (2017). For the simulation depicted in Fig.…”

Section: Mathematical Framework and Examplesmentioning

confidence: 99%

Renewal Reward Perspective on Linear Switching Diffusion Systems in Models of Intracellular Transport

et al. 2020

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In many biological systems, the movement of individual agents is characterized having multiple qualitatively distinct behaviors that arise from a variety of biophysical states. For example, in cells the movement of vesicles, organelles, and other intracellular cargo is affected by their binding to and unbinding from cytoskeletal filaments such as microtubules through molecular motor proteins. A typical goal of theoretical or numerical analysis of models of such systems is to investigate effective transport properties and their dependence on model parameters. While the effective velocity of particles undergoing switching diffusion dynamics is often easily characterized in terms of the long-time fraction of time that particles spend in each state, the calculation of the effective diffusivity is more complicated because it cannot be expressed simply in terms of a statistical average of the particle transport state at one moment of time. However, it is common that these systems are regenerative, in the sense that they can be decomposed into independent cycles marked by returns to a base state. Using decompositions of this kind, we calculate effective transport properties by computing the moments of the dynamics within each cycle and then applying renewal reward theory. This method provides a useful alternative large-time analysis to direct homogenization for linear advection–reaction–diffusion partial differential equation models. Moreover, it applies to a general class of semi-Markov processes and certain stochastic differential equations that arise in models of intracellular transport. Applications of the proposed renewal reward framework are illustrated for several case studies such as mRNA transport in developing oocytes and processive cargo movement by teams of molecular motor proteins.

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KymoAnalyzer: a software tool for the quantitative analysis of intracellular transport in neurons

Cited by 88 publications

References 43 publications

Limiting Neuronal Nogo Receptor 1 Signaling during Experimental Autoimmune Encephalomyelitis Preserves Axonal Transport and Abrogates Inflammatory Demyelination

Limiting Neuronal Nogo Receptor 1 Signaling during Experimental Autoimmune Encephalomyelitis Preserves Axonal Transport and Abrogates Inflammatory Demyelination

Motor reattachment kinetics play a dominant role in multimotor-driven cargo transport

Renewal Reward Perspective on Linear Switching Diffusion Systems in Models of Intracellular Transport

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