Polarized localization of kinesin-1 and RIC-7 drives axonal mitochondria anterograde transport.

Mitochondria transport is crucial for axonal mitochondria distribution and is mediated by kinesin-1-based anterograde and dynein-based retrograde motor complexes. While Miro and Milton/TRAK were identified as key adaptors between mitochondria and kinesin-1, recent studies suggest the presence of additional mechanisms. In C. elegans, ric-7 is the only single gene described so far, other than kinesin-1, that is absolutely required for axonal mitochondria localization. Using CRISPR engineering in C. elegans, we find that Miro is important but is not essential for anterograde traffic, whereas it is required for retrograde traffic. Both the endogenous RIC-7 and kinesin-1 act at the leading end to transport mitochondria anterogradely. RIC-7 binding to mitochondria requires its N-terminal domain and partially relies on MIRO-1, whereas RIC-7 accumulation at the leading end depends on its disordered region, kinesin-1, and metaxin2. We conclude that transport complexes containing kinesin-1 and RIC-7 polarize at the leading edge of mitochondria and are required for anterograde axonal transport in C. elegans.

Optimizing Visualization of Axonal Transport of Endogenous Cargo by Fluorescence Microscopy in Living Caenorhabditis elegans.

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. Consequently, loss of axonal transport impairs neuronal growth and function. Studying axonal transport therefore improves our understanding of neuronal cell biology. With recent improvements in CRISPR Cas9 genome editing, endogenous labeling of axonal cargos has become accessible, enabling to move beyond ectopic expression-based visualization of transport. However, endogenous labeling often comes at the cost of low signal intensity and necessitates optimization strategies to obtain robust data. Here, we describe a protocol to optimize the visualization of axonal transport by discussing acquisition parameters and a bleaching approach to improve the signal of endogenous labeled cargo over diffuse cytoplasmic background. We apply our protocol to optimize the visualization of synaptic vesicle precursors (SVPs) labeled by green fluorescent protein (GFP)-tagged RAB-3 to highlight how fine-tuning acquisition parameters can improve the analysis of endogenously labeled axonal cargo in Caenorhabditis elegans (C. elegans).

A kinesin-1 adaptor complex controls bimodal slow axonal transport of spectrin in Caenorhabditis elegans.

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.

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

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.

Polarized localization of kinesin-1 and RIC-7 drives axonal mitochondria anterograde transport.

Mitochondria transport is crucial for mitochondria distribution in axons and is mediated by kinesin-1-based anterograde and dynein-based retrograde motor complexes. While Miro and Milton/TRAK were identified as key adaptors between mitochondria and kinesin-1, recent studies suggest the presence of additional mechanisms. In C. elegans, ric-7 is the only single gene described so far, other than kinesin-1, that is absolutely required for axonal mitochondria localization. Using CRISPR engineering in C. elegans, we find that Miro is important but is not essential for anterograde traffic, whereas it is required for retrograde traffic. Both the endogenous RIC-7 and kinesin-1 act at the leading end to transport mitochondria anterogradely. RIC-7 recruitment to mitochondria requires its N-terminal domain and partially relies on MIRO-1, whereas RIC-7 accumulation at the leading end depends on its disordered region, kinesin-1 and metaxin2. We conclude that polarized transport complexes containing kinesin-1 and RIC-7 form at the leading edge of mitochondria, and that these complexes are required for anterograde axonal transport.

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

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.

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

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 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 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. 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 ebp-1 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 ebp-1 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.

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

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.

InterSEPTIN' Kinesins in Dendrites.

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.

Establishing Neuronal Polarity with Environmental and Intrinsic Mechanisms.

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.

Local inhibition of microtubule dynamics by dynein is required for neuronal cargo distribution.

Abnormal axonal transport is associated with neuronal disease. We identified a role for DHC-1, the C. elegans dynein heavy chain, in maintaining neuronal cargo distribution. Surprisingly, this does not involve dynein's role as a retrograde motor in cargo transport, hinging instead on its ability to inhibit microtubule (MT) dynamics. Neuronal MTs are highly static, yet the mechanisms and functional significance of this property are not well understood. In disease-mimicking dhc-1 alleles, excessive MT growth and collapse occur at the dendrite tip, resulting in the formation of aberrant MT loops. These unstable MTs act as cargo traps, leading to ectopic accumulations of cargo and reduced availability of cargo at normal locations. Our data suggest that an anchored dynein pool interacts with plus-end-out MTs to stabilize MTs and allow efficient retrograde transport. These results identify functional significance for neuronal MT stability and suggest a mechanism for cellular dysfunction in dynein-linked disease.

Microtubule Organization Determines Axonal Transport Dynamics.

Axonal microtubule (MT) arrays are the major cytoskeleton substrate for cargo transport. How MT organization, i.e., polymer length, number, and minus-end spacing, is regulated and how it impinges on axonal transport are unclear. We describe a method for analyzing neuronal MT organization using light microscopy. This method circumvents the need for electron microscopy reconstructions and is compatible with live imaging of cargo transport and MT dynamics. Examination of a C. elegans motor neuron revealed how age, MT-associated proteins, and signaling pathways control MT length, minus-end spacing, and coverage. In turn, MT organization determines axonal transport progression: cargoes pause at polymer termini, suggesting that switching MT tracks is rate limiting for efficient transport. Cargo run length is set by MT length, and higher MT coverage correlates with shorter pauses. These results uncover the principles and mechanisms of neuronal MT organization and its regulation of axonal cargo transport.

Cellular and molecular mechanisms of synaptic specificity.

Precise connectivity in neuronal circuits is a prerequisite for proper brain function. The dauntingly complex environment encountered by axons and dendrites, even after navigation to their target area, prompts the question of how specificity of synaptic connections arises during development. We review developmental strategies and molecular mechanisms that are used by neurons to ensure their precise matching of pre- and postsynaptic elements. The emerging theme is that each circuit uses a combination of simple mechanisms to achieve its refined, often complex connectivity pattern. At increasing levels of resolution, from lamina choice to subcellular targeting, similar signaling concepts are reemployed to narrow the choice of potential matches. Temporal control over synapse development and synapse elimination further ensures the specificity of connections in the nervous system.

Sequential activation of ETS proteins provides a sustained transcriptional response to EGFR signaling.

How signal transduction, which is dynamic and fluctuating by nature, is converted into a stable trancriptional response, is an unanswered question in developmental biology. Two ETS-domain transcription factors encoded by the pointed (pnt) locus, PntP1 and PntP2, are universal downstream mediators of EGFR-based signaling in Drosophila. Full disruption of pnt function in developing eye imaginal discs reveals a photoreceptor recruitment phenotype, in which only the R8 photoreceptor cell type is specified within ommatidia. Specific disruption of either pntP1 or pntP2 resulted in the same R8-only phenotype, demonstrating that both Pnt isoforms are essential for photoreceptor recruitment. We show that the two Pnt protein forms are activated in a sequential manner within the EGFR signaling pathway: MAPK phosphorylates and activates PntP2, which in turn induces pntP1 transcription. Once expressed, PntP1 is constitutively active and sufficient to induce target genes essential for photoreceptor development. Pulse-chase experiments indicate that PntP1 is stable for several hours in the eye disc. Sequential ETS-protein recruitment therefore allows sustained induction of target genes, beyond the transient activation of EGFR.

Versatility of EGF receptor ligand processing in insects.

Processing of EGF-family ligands is an essential step in triggering the EGF receptor pathway, which fulfills a diverse set of roles during development and tissue maintenance. We describe a mechanism of ligand processing which is unique to insects, and possibly to other invertebrates. This mechanism relies on ligand precursor trafficking from the ER by a chaperone, Star (S), and precursor cleavage by Rhomboids, a family of intra-membrane protease. Remarkably, the ability of Rhomboids to cleave S as well, endows the pathway with additional diversity. Rhomboid isoforms which also reside in the ER inactivate the chaperone before any ligand was trafficked, thus significantly reducing the level of ligand that will eventually be processed and secreted. ER localization also serves as a critical feature in trafficking the entire ligand-processing machinery to axonal termini, as the ER extends throughout the axon. Finally, examination of diverse species of insects demonstrates the evolution of chaperone cleavability, indicating that the primordial processing machinery could support long-range signaling by the ligand. Altering the intracellular localization of critical components of a conserved signaling cassette therefore provides an evolutionary mechanism for modulation of signaling levels, and diversification of the biological settings where the pathway functions.

Polarized secretion of Drosophila EGFR ligand from photoreceptor neurons is controlled by ER localization of the ligand-processing machinery.

The release of signaling molecules from neurons must be regulated, to accommodate their highly polarized structure. In the developing Drosophila visual system, photoreceptor neurons secrete the epidermal growth factor receptor ligand Spitz (Spi) from their cell bodies, as well as from their axonal termini. Here we show that subcellular localization of Rhomboid proteases, which process Spi, determines the site of Spi release from neurons. Endoplasmic reticulum (ER) localization of Rhomboid 3 is essential for its ability to promote Spi secretion from axons, but not from cell bodies. We demonstrate that the ER extends throughout photoreceptor axons, and show that this feature facilitates the trafficking of the Spi precursor, the ligand chaperone Star, and Rhomboid 3 to axonal termini. Following this trafficking step, secretion from the axons is regulated in a manner similar to secretion from cell bodies. These findings uncover a role for the ER in trafficking proteins from the neuronal cell body to axon terminus.

Generation of distinct signaling modes via diversification of the Egfr ligand-processing cassette.

Egfr ligand processing in Drosophila involves trafficking of the ligand precursor by the chaperone Star from the endoplasmic reticulum (ER) to a secretory compartment, where the precursor is cleaved by the intramembrane protease Rhomboid. Some of the Drosophila Rhomboids also reside in the ER, where they attenuate signaling by premature cleavage of Star. The genome of the flour beetle Tribolium castaneum contains a single gene for each of the ligand-processing components, providing an opportunity to assess the regulation and impact of a simplified ligand-processing cassette. We find that the central features of ligand retention, trafficking by the chaperone and cleavage by Rhomboid have been conserved. The single Rhomboid is localized to both ER and secretory compartments. However, we show that Tribolium Star is refractive to Rhomboid cleavage. Consequently, this ligand-processing system effectively mediates long-range Egfr activation in the Tribolium embryonic ventral ectoderm, despite ER localization of Rhomboid. Diversification of the Egfr signaling pathway appears to have coupled gene duplication events with modulation of the biochemical properties and subcellular localization patterns of Rhomboid proteases and their substrates.

Drosophila EGFR signalling is modulated by differential compartmentalization of Rhomboid intramembrane proteases.

We explore the role of differential compartmentalization of Rhomboid (Rho) proteases that process the Drosophila EGF receptor ligands, in modulating the amount of secreted ligand and consequently the level of EGF receptor (EGFR) activation. The mSpitz ligand precursor is retained in the ER, and is trafficked by the chaperone Star to a late compartment of the secretory pathway, where Rho-1 resides. This work demonstrates that two other Rho proteins, Rho-2 and Rho-3, which are expressed in the germ line and in the developing eye, respectively, cleave the Spitz precursor and Star already in the ER, in addition to their activity in the late compartment. This property attenuates EGFR activation, primarily by compromising the amount of chaperone that can productively traffic the ligand precursor to the late compartment, where cleavage and subsequent secretion take place. These observations identify changes in intracellular compartment localization of Rho proteins as a basis for signal attenuation, in tissues where EGFR activation must be highly restricted in space and time.

Rhomboid cleaves Star to regulate the levels of secreted Spitz.

Intracellular trafficking of the precursor of Spitz (Spi), the major Drosophila EGF receptor (EGFR) ligand, is facilitated by the chaperone Star, a type II transmembrane protein. This study identifies a novel mechanism for modulating the activity of Star, thereby influencing the levels of active Spi ligand produced. We demonstrate that Star can efficiently traffic Spi even when present at sub-stoichiometric levels, and that in Drosophila S(2)R(+) cells, Spi is trafficked from the endoplasmic reticulum to the late endosome compartment, also enriched for Rhomboid, an intramembrane protease. Rhomboid, which cleaves the Spi precursor, is now shown to also cleave Star within its transmembrane domain both in cell culture and in flies, expanding the repertoire of known Rhomboid substrates to include both type I and type II transmembrane proteins. Cleavage of Star restricts the amount of Spi that is trafficked, and may explain the exceptional dosage sensitivity of the Star locus in flies.

The apical determinants aPKC and dPatj regulate Frizzled-dependent planar cell polarity in the Drosophila eye.

Planar cell polarity (PCP) is a common feature of many vertebrate and invertebrate epithelia and is perpendicular to their apical/basal (A/B) polarity axis. While apical localization of PCP determinants such as Frizzled (Fz1) is critical for their function, the link between A/B polarity and PCP is poorly understood. Here, we describe a direct molecular link between A/B determinants and Fz1-mediated PCP establishment in the Drosophila eye. We demonstrate that dPatj binds the cytoplasmic tail of Fz1 and propose that it recruits aPKC, which in turn phosphorylates and inhibits Fz1. Accordingly, components of the aPKC complex and dPatj produce PCP defects in the eye. We also show that during PCP signaling, aPKC and dPatj are downregulated, while Bazooka is upregulated, suggesting an antagonistic effect of Bazooka on dPatj/aPKC. We propose a model whereby the dPatj/aPKC complex regulates PCP by inhibiting Fz1 in cells where it should not be active.