Imaging Intracellular Trafficking in Neurons of C. elegans

Nadiminti, Sravanthi S P; Koushika, Sandhya P.

doi:10.1007/978-1-0716-1990-2_27

Cited by 6 publications

(5 citation statements)

References 67 publications

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“…All analysis was done using FIJI (Schindelin et al, 2012). For detailed methods, please refer to (Nadiminti and Koushika, 2022).…”

Section: Discussionmentioning

confidence: 99%

“…Fraction of GFP-positive vesicles co-migrating with RFP-positive vesicles = For detailed methods, please refer to (Nadiminti and Koushika, 2022). (iii) Quantitation of penetrance of CTNS-1 puncta that exit into PLM neurites: For each genotype, at least 30 animals were annotated to observe the extent of CTNS-1 (or RAB-7 or LMP-1) presence in the PLM major neurite.…”

Section: Methodsmentioning

confidence: 99%

“…For detailed methods, please refer to (Nadiminti and Koushika, 2022). (vi) Microtubule polarity: Kymographs were generated from live movies of EBP-2::GFP in the axonal and anterior dendritic regions of the PVD neuron imaged at 3 fps.…”

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confidence: 99%

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Active zone protein SYD-2/Liprin-α acts downstream of LRK-1/LRRK2 to regulate polarized trafficking of synaptic vesicle precursors through clathrin adaptor protein complexes

Nadiminti

Dixit

Ratnakaran

et al. 2023

Preprint

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Synaptic vesicle proteins (SVps) are thought to travel in heterogeneous carriers dependent on the motor UNC-104/KIF1A. In C. elegans neurons, we found that some SVps are transported along with lysosomal proteins by the motor UNC-104/KIF1A. LRK-1/LRRK2 and the clathrin adaptor protein complex AP-3 are critical for the separation of lysosomal proteins from SVp transport carriers. In lrk-1 mutants, both SVp carriers and SVp carriers containing lysosomal proteins are independent of UNC-104, suggesting that LRK-1 plays a key role in ensuring UNC-104-dependent transport of SVps. Additionally, LRK-1 likely acts upstream of the AP-3 complex and regulates the membrane localization of AP-3. The action of AP-3 is necessary for the active zone protein SYD-2/Liprin-α to facilitate the transport of SVp carriers. In the absence of the AP-3 complex, SYD-2/Liprin-α acts with UNC-104 to instead facilitate the transport of SVp carriers containing lysosomal proteins. We further show that the mistrafficking of SVps into the dendrite in lrk-1 and apb-3 mutants depends on SYD-2, likely by regulating the recruitment of the AP-1/UNC-101. We propose that SYD-2 acts in concert with both the AP-1 and AP-3 complexes to ensure polarized trafficking of SVps.

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“…All analysis was done using FIJI (Schindelin et al, 2012). For detailed methods, please refer to (Nadiminti and Koushika, 2022).…”

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Active zone protein SYD-2/Liprin-α acts downstream of LRK-1/LRRK2 to regulate polarized trafficking of synaptic vesicle precursors through clathrin adaptor protein complexes

Nadiminti

Dixit

Ratnakaran

et al. 2023

Preprint

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“…Chai et al, 2012) and subcellular processes in vivo over time (e.g. Nadiminti and Koushika, 2022), monitoring the concentration and localization of cellular metabolites over time (e.g. Venkatachalam et al, 2016) and selective labeling for biochemical studies (e.g.…”

Section: Introductionmentioning

confidence: 99%

Parameters that influence bipartite reporter system expression inC. elegans

Knoebel,

Brinck,

Nonet

2024

Preprint

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The development of bipartite reporter systems inC. eleganshas lagged by more than a decade behind its adoption in Drosophila, the other invertebrate model commonly used to dissect biological mechanisms. Here, we characterize many parameters that influence expression in recently developedC. elegansbipartite systems. We examine how DNA binding site number and spacing influence expression and characterize how these expression parameters vary in distinct tissue types. Furthermore, we examine how both basal promoters and 3’ UTR influence the specificity and level of expression. These studies provide both a framework for the rational design of driver and reporter transgenes as well as molecular and genetic tools for the creation, characterization, and optimization of bipartite system components for expression in other cell types.

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“…To study transport in a more physiological environment, several in vivo methods have also been established to enable tracking of individual axonal cargoes [ 17 ]; for example, trafficking can be imaged in Drosophila larvae and adults [ 18 , 19 ], different zebrafish [ 20 , 21 ] and Caenorhabditis elegans [ 22 ] neurons, as well as in rodent sciatic nerves [ 23 ], spinal cord and dorsal roots [ 24 ], retinal ganglion cells [ 25 ] and the brain [ 26 ]. Considering the selective vulnerability of neurons across different neurological diseases, designing complementary intravital imaging methods to allow comparative transport analyses across neuronal subtypes and anatomically distinct regions will be a useful addition to the axonal transport toolkit.…”

Section: Introductionmentioning

confidence: 99%

In vivo imaging of axonal transport in peripheral nerves of rodent forelimbs

2023

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Axonal transport is the essential process by which neurons actively traffic a variety of cargoes between the cell soma and axon terminals. Accordingly, dysfunctional axonal transport is linked to many nervous system conditions. Therefore, being able to image and quantify this dynamic process in live neurons of animal disease models is beneficial for understanding neuropathology and testing new therapies at the preclinical level. As such, intravital approaches have been developed to assess cargo movement in the hindlimb sciatic nerves of live, anaesthetised mice. Here, we describe an adapted method for in vivo imaging of axonal transport in intact median and ulnar nerves of the rodent forelimb. Injection of a fluorescently labelled and non-toxic fragment of tetanus neurotoxin (HCT) into the mouse forepaw permits the identification of signalling endosomes in intact axons of median and ulnar nerves. Through immunofluorescent analysis of forelimb lumbrical muscles and median/ulnar nerves, we confirmed that HCT is taken up at motor nerve terminals and predominantly locates to motor axons. We then showed that the baseline trafficking of signalling endosomes is similar between the median/ulnar nerves and the sciatic nerve in adult wild-type mice. Importantly, this adapted method can be readily tailored for assessment of additional cargoes, such as mitochondria. By measuring transport in forelimb and hindlimb nerves, comparative anatomical and functional analyses can be performed in rodent disease models to aid our understanding of peripheral nerve disease pathogenesis and response to injury.

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Imaging Intracellular Trafficking in Neurons of C. elegans

Cited by 6 publications

References 67 publications

Active zone protein SYD-2/Liprin-α acts downstream of LRK-1/LRRK2 to regulate polarized trafficking of synaptic vesicle precursors through clathrin adaptor protein complexes

Active zone protein SYD-2/Liprin-α acts downstream of LRK-1/LRRK2 to regulate polarized trafficking of synaptic vesicle precursors through clathrin adaptor protein complexes

Parameters that influence bipartite reporter system expression inC. elegans

In vivo imaging of axonal transport in peripheral nerves of rodent forelimbs

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