Visualizing Vesicle-Bound Kinesins in Cultured Hippocampal Neurons

Garbouchian, Alex; Bentley, Marvin

doi:10.1007/978-1-0716-1990-2_12

Cited by 9 publications

(13 citation statements)

References 43 publications

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“…However, the comparable velocities between KAP and KIF3AB/KIF3AC suggests that the motor complexes are not substantially impaired in their ability to move organelles. Furthermore, previous experiments with kinesin tail constructs did not find any substantial change in the trafficking behavior of cargo-labeled vesicles (Jenkins et al, 2012;Yang et al, 2019;Montgomery et al, 2022;Frank et al, 2020;. A third interpretation is that KAP binding to organelles is more stable than KIF3AB or KIF3AC binding to KAP.…”

Section: Kif3ab Kif3ac and Kap Organelles Exhibit Similar Cellular Di...mentioning

confidence: 78%

“…Because KIF3B and KIFC both bind KAP, they may interact with some of the same neuronal organelle populations. We recently developed techniques for visualizing organelle-bound kinesins in live cells (Montgomery et al, 2022; Yang et al, 2019; Frank et al, 2020). Improved organelle labeling was achieved by expressing the GFP-tagged kinesin tail domains, which mediate organelle binding.…”

Section: Resultsmentioning

confidence: 99%

“…The halotag enzyme itself is not fluorescent and becomes visible only after covalent binding to a fluorescent ligand. Bright organic dyes have substantially more favorable fluorescence characteristics than fluorescent proteins (Grimm et al, 2015; Xia et al, 2013) and enhance detection of organelle-bound proteins (Montgomery et al, 2022; Frank et al, 2020; Yang et al, 2019). To use this approach, we generated halo-tagged KIF3B and KIF3C tail constructs (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Because KIF3B and KIFC both bind KAP, they may interact with some of the same neuronal organelle populations. We recently developed techniques for visualizing organellebound kinesins in live cells (Montgomery et al, 2022;Yang et al, 2019;Frank et al, 2020).…”

Section: Kif3ab Kif3ac and Kap Organelles Exhibit Similar Cellular Di...mentioning

confidence: 99%

“…Moreover, the KAP binding site for both heterodimers is localized in their C-terminal disordered domains. Second, we expressed fluorescent kinesin tail domain in cultured hippocampal neurons to analyze the localization and trafficking parameters of organelle-bound kinesins (Montgomery et al, 2022; Yang et al, 2019). With this approach, we found that KIF3AB, KIF3AC, and KAP bind the same neuronal organelles.…”

Section: Introductionmentioning

confidence: 99%

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KAP is the neuronal organelle adaptor for Kinesin-2 KIF3AB and KIF3AC

Garbouchian

Gilbert

Bentley

2022

Preprint

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Kinesin-driven organelle transport is crucial for neuron development and maintenance, yet the mechanisms by which kinesins specifically bind their organelle cargoes remain undefined. In contrast to other transport kinesins, the neuronal function and specific organelle adaptors of heterodimeric Kinesin-2 family members KIF3AB and KIF3AC remain unknown. We developed a novel microscopy-based assay to define protein-protein interactions in intact neurons. The experiments found that KIF3AB and KIF3AC both bind KAP. These interactions are mediated by the distal C-terminal tail regions and not the coiled-coil domain. We used live-cell imaging in cultured hippocampal neurons to define the localization and trafficking parameters of KIF3AB and KIF3AC organelle populations. We discovered that KIF3AB/KAP and KIF3AC/KAP bind the same organelle populations and defined their transport parameters in axons and dendrites. The results also show that ~12% of KIF3 organelles contain the RNA binding protein, adenomatous polyposis coli. These data point towards a model in which KIF3AB and KIF3AC use KAP as their neuronal organelle adaptor and that these kinesins mediate transport of a range of organelles.

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Section: Kif3ab Kif3ac and Kap Organelles Exhibit Similar Cellular Di...mentioning

confidence: 78%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Kif3ab Kif3ac and Kap Organelles Exhibit Similar Cellular Di...mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

KAP is the neuronal organelle adaptor for Kinesin-2 KIF3AB and KIF3AC

Garbouchian

Gilbert

Bentley

2022

Preprint

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High-Resolution Imaging of Mitochondria and Mitochondrial Nucleoids in Differentiated SH-SY5Y Cells

Annuario

Vagnoni

2022

Methods in Molecular Biology

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Mitochondria are highly dynamic organelles which form intricate networks with complex dynamics. Mitochondrial transport and distribution are essential to ensure proper cell function, especially in cells with an extremely polarised morphology such as neurons. A layer of complexity is added when considering mitochondria have their own genome, packaged into nucleoids. Major mitochondrial morphological transitions, for example mitochondrial division, often occur in conjunction with mitochondrial DNA (mtDNA) replication and changes in the dynamic behaviour of the nucleoids. However, the relationship between mtDNA dynamics and mitochondrial motility in the processes of neurons has been largely overlooked. In this chapter, we describe a method for live imaging of mitochondria and nucleoids in differentiated SH-SY5Y cells by instant structured illumination microscopy (iSIM). We also include a detailed protocol for the differentiation of SH-SY5Y cells into cells with a pronounced neuronal-like morphology and show examples of coordinated mitochondrial and nucleoid motility in the long processes of these cells.

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Detailed Imaging of Mitochondrial Transport and Precise Manipulation of Mitochondrial Function with Genetically Encoded Photosensitizers in Adult Drosophila Neurons

Mattedi

Chennell

Vagnoni

2022

Methods in Molecular Biology

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Precise distribution of mitochondria is essential for maintaining neuronal homeostasis. Although detailed mechanisms governing the transport of mitochondria have emerged, it is still poorly understood how the regulation of transport is coordinated in space and time within the physiological context of an organism. How alteration in mitochondrial functionality may trigger changes in organellar dynamics also remains unclear in this context. Therefore, the use of genetically encoded tools to perturb mitochondrial functionality in real time would be desirable. Here we describe methods to interfere with mitochondrial function with high spatiotemporal precision with the use of photosensitizers in vivo in the intact wing nerve of adult Drosophila. We also provide details on how to visualize the transport of mitochondria and to improve the quality of the imaging to attain super-resolution in this tissue.

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Visualizing Vesicle-Bound Kinesins in Cultured Hippocampal Neurons

Cited by 9 publications

References 43 publications

KAP is the neuronal organelle adaptor for Kinesin-2 KIF3AB and KIF3AC

KAP is the neuronal organelle adaptor for Kinesin-2 KIF3AB and KIF3AC

High-Resolution Imaging of Mitochondria and Mitochondrial Nucleoids in Differentiated SH-SY5Y Cells

Detailed Imaging of Mitochondrial Transport and Precise Manipulation of Mitochondrial Function with Genetically Encoded Photosensitizers in Adult Drosophila Neurons

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