optimizing-visualization-axonal-transport-endogenous-cargo

Optimizing Visualization of Axonal Transport of Endogenous Cargo by Fluorescence Microscopy in Living <em>Caenorhabditis elegans</em>

Axonal transport is a prerequisite to deliver axonal proteins from their site of synthesis in the neuronal cell body to their destination in the axon. ...

Yale School of Medicine

Neurons are among the most morphologically complex cells. A distinction between two compartments, axon and dendrite, generates cellular domains that differ in membrane composition and cytoskeletal structure, and sets the platform on which morphogens, transcription programs, and synaptic activity sculpt neuronal form. The establishment of this distinction, called Neuronal Polarity, entails interpreting spatial and intrinsic cues and converting them to cytoskeletal rearrangements that give rise to axons and dendrites. Hence, this early developmental event underpins the future functional properties of the neuron to receive and transmit information. Here we review the current understanding of developmental cues and cell biological mechanisms that establish polarity in newborn neurons, synthesizing information from vertebrate and invertebrate model systems.

Establishing Neuronal Polarity with Environmental and Intrinsic Mechanisms.

Cargo transport to axons and dendrites is essential for maintaining neuronal polarity and function. In this issue of Developmental Cell, Karasmanis et al. (2018) identify a septin-SEPT9-in differentially regulating the motility of two kinesin motors, thereby controlling cargo entry into dendrites.

InterSEPTIN' Kinesins in Dendrites.

Synapse formation is locally determined by transmembrane proteins, yet synaptic material is synthesized remotely and undergoes processive transport in axons. How local synaptogenic signals intercept synaptic cargo in transport to promote its delivery and synapse formation is unknown. We found that the control of synaptic cargo delivery at microtubule (MT) minus ends mediates pro- and anti-synaptogenic activities of presynaptic neurexin and frizzled in C. elegans and identified the atypical kinesin VAB-8/KIF26 as a key molecule in this process. VAB-8/KIF26 levels at synaptic MT minus ends are controlled by frizzled and neurexin; loss of VAB-8 mimics neurexin mutants or frizzled hyperactivation, and its overexpression can rescue synapse loss in these backgrounds. VAB-8/KIF26 is required for the synaptic localization of other minus-end proteins and promotes the pausing of retrograde transport to allow delivery to synapses. Consistently, reducing retrograde transport rescues synapse loss in vab-8 and neurexin mutants. These results uncover a mechanistic link between synaptogenic signaling and axonal transport.

Neurexin and frizzled intercept axonal transport at microtubule minus ends to control synapse formation.

Axonal transport is key to neuronal function. Efficient transport requires specific motor-cargo association in the soma, yet the mechanisms regulating this early step remain poorly understood. We found that EBP-1, the  ortholog of the canonical microtubule end binding protein EB1, promotes the specific association between kinesin-3/KIF1A/UNC-104 and Dense Core Vesicles (DCVs) prior to their axonal delivery. Using single-neuron,  labelling of endogenous cargo and EBs, we observed reduced axonal abundance and reduced secretion of DCV cargo, but not other KIF1A/UNC-104 cargo, in  mutants. This reduction could be traced back to fewer exit events from the cell body, where EBP-1 colocalized with the DCV sorting machinery at the trans Golgi, suggesting that this is the site of EBP-1 function. In addition to its microtubule binding CH domain, mammalian EB1 interacted with mammalian KIF1A in an EBH domain dependent manner, and expression of mammalian EB1 or the EBH domain was sufficient to rescue DCV transport in  mutants. Our results suggest a model in which kinesin-3 binding and microtubule binding by EBP-1 cooperate to transiently enrich the motor near sites of DCV biogenesis to promote motor-cargo association. In support of this model, tethering either EBP-1 or a kinesin-3 KIF1A/UNC-104 interacting domain from an unrelated protein to the Golgi restored the axonal abundance of DCV proteins in  mutants. These results uncover an unexpected role for a microtubule associated protein and provide insight into how specific kinesin-3 cargo are delivered to the axon.

End Binding protein 1 promotes specific motor-cargo association in the cell body prior to axonal delivery of Dense Core Vesicles.

Cell invasion through basement membrane (BM) barriers is important in development, immune function and cancer progression. As invasion through BM is often stochastic, capturing gene expression profiles of actively invading cells in vivo remains elusive. Using the stereotyped timing of Caenorhabditis elegans anchor cell (AC) invasion, we generated an AC transcriptome during BM breaching. Through a focused RNAi screen of transcriptionally enriched genes, we identified new invasion regulators, including translationally controlled tumor protein (TCTP). We also discovered gene enrichment of ribosomal proteins. AC-specific RNAi, endogenous ribosome labeling and ribosome biogenesis analysis revealed that a burst of ribosome production occurs shortly after AC specification, which drives the translation of proteins mediating BM removal. Ribosomes also enrich near the AC endoplasmic reticulum (ER) Sec61 translocon and the endomembrane system expands before invasion. We show that AC invasion is sensitive to ER stress, indicating a heightened requirement for translation of ER-trafficked proteins. These studies reveal key roles for ribosome biogenesis and endomembrane expansion in cell invasion through BM and establish the AC transcriptome as a resource to identify mechanisms underlying BM transmigration.

The Caenorhabditis elegans anchor cell transcriptome: ribosome biogenesis drives cell invasion through basement membrane.

Axonal transport is key to neuronal function. Efficient transport requires specific motor-cargo association in the soma, yet the mechanisms regulating this early step remain poorly understood. We found that EBP-1, the C. elegans ortholog of the canonical-microtubule-end-binding protein EB1, promotes the specific association between kinesin-3/KIF1A/UNC-104 and dense core vesicles (DCVs) prior to their axonal delivery. Using single-neuron, in vivo labeling of endogenous cargo and EBs, we observed reduced axonal abundance and reduced secretion of DCV cargo, but not other KIF1A/UNC-104 cargoes, in ebp-1 mutants. This reduction could be traced back to fewer exit events from the cell body, where EBP-1 colocalized with the DCV sorting machinery at the trans Golgi, suggesting that this is the site of EBP-1 function. EBP-1 calponin homology (CH) domain was required for directing microtubule growth on the Golgi, and mammalian EB1 interacted with KIF1A in an EBH-domain-dependent manner. Loss- and gain-of-function experiments suggest a model in which both kinesin-3 binding and guidance of microtubule growth at the trans Golgi by EBP-1 promote motor-cargo association at sites of DCV biogenesis. In support of this model, tethering either EBP-1 or a kinesin-3/KIF1A/UNC-104-interacting domain from an unrelated protein to the Golgi restored the axonal abundance of DCV proteins in ebp-1 mutants. These results uncover an unexpected role for a microtubule-associated protein and provide insights into how specific kinesin-3 cargo is delivered to the axon.

End-binding protein 1 promotes specific motor-cargo association in the cell body prior to axonal delivery of dense core vesicles.

An actin-spectrin lattice, the membrane periodic skeleton (MPS), protects axons from breakage. MPS integrity relies on spectrin delivery via slow axonal transport, a process that remains poorly understood. We designed a probe to visualize endogenous spectrin dynamics at single-axon resolution in vivo. Surprisingly, spectrin transport is bimodal, comprising fast runs and movements that are 100-fold slower than previously reported. Modeling and genetic analysis suggest that the two rates are independent, yet both require kinesin-1 and the coiled-coil proteins UNC-76/FEZ1 and UNC-69/SCOC, which we identify as spectrin-kinesin adaptors. Knockdown of either protein led to disrupted spectrin motility and reduced distal MPS, and UNC-76 overexpression instructed excessive transport of spectrin. Artificially linking spectrin to kinesin-1 drove robust motility but inefficient MPS assembly, whereas impairing MPS assembly led to excessive spectrin transport, suggesting a balance between transport and assembly. These results provide insight into slow axonal transport and MPS integrity.

Article	Year
Establishing Neuronal Polarity with Environmental and Intrinsic Mechanisms. Neuron\| PubMed ID: 29096077	2017
InterSEPTIN' Kinesins in Dendrites. Developmental cell\| PubMed ID: 30016616	2018
Neurexin and frizzled intercept axonal transport at microtubule minus ends to control synapse formation. Developmental cell\| PubMed ID: 35809561	2022
End Binding protein 1 promotes specific motor-cargo association in the cell body prior to axonal delivery of Dense Core Vesicles. bioRxiv : the preprint server for biology\| PubMed ID: 36711860	2023
The Caenorhabditis elegans anchor cell transcriptome: ribosome biogenesis drives cell invasion through basement membrane. Development (Cambridge, England)\| PubMed ID: 37039075	2023
End-binding protein 1 promotes specific motor-cargo association in the cell body prior to axonal delivery of dense core vesicles. Current biology : CB\| PubMed ID: 37586371	2023
A kinesin-1 adaptor complex controls bimodal slow axonal transport of spectrin in Caenorhabditis elegans. Developmental cell\| PubMed ID: 37751746	2023

Shaul Yogev

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