Imaging the Polarized Sorting of Proteins from the Golgi Complex in Live Neurons

Farı́as, Ginny G.; Britt, Dylan; Bonifacino, Juan S.

doi:10.1007/978-1-4939-6463-5_2

Cited by 20 publications

(18 citation statements)

References 19 publications

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“…Primary hippocampal neurons were prepared from 18.5-day-old (E18.5) WT and AP-4 ε KO mouse embryos as previously described [ 67 ]. Cortical neurons were prepared by an adaptation of the protocol for hippocampal neurons.…”

Section: Methodsmentioning

confidence: 99%

Altered distribution of ATG9A and accumulation of axonal aggregates in neurons from a mouse model of AP-4 deficiency syndrome

et al. 2018

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The hereditary spastic paraplegias (HSP) are a clinically and genetically heterogeneous group of disorders characterized by progressive lower limb spasticity. Mutations in subunits of the heterotetrameric (ε-β4-μ4-σ4) adaptor protein 4 (AP-4) complex cause an autosomal recessive form of complicated HSP referred to as “AP-4 deficiency syndrome”. In addition to lower limb spasticity, this syndrome features intellectual disability, microcephaly, seizures, thin corpus callosum and upper limb spasticity. The pathogenetic mechanism, however, remains poorly understood. Here we report the characterization of a knockout (KO) mouse for the AP4E1 gene encoding the ε subunit of AP-4. We find that AP-4 ε KO mice exhibit a range of neurological phenotypes, including hindlimb clasping, decreased motor coordination and weak grip strength. In addition, AP-4 ε KO mice display a thin corpus callosum and axonal swellings in various areas of the brain and spinal cord. Immunohistochemical analyses show that the transmembrane autophagy-related protein 9A (ATG9A) is more concentrated in the trans-Golgi network (TGN) and depleted from the peripheral cytoplasm both in skin fibroblasts from patients with mutations in the μ4 subunit of AP-4 and in various neuronal types in AP-4 ε KO mice. ATG9A mislocalization is associated with increased tendency to accumulate mutant huntingtin (HTT) aggregates in the axons of AP-4 ε KO neurons. These findings indicate that the AP-4 ε KO mouse is a suitable animal model for AP-4 deficiency syndrome, and that defective mobilization of ATG9A from the TGN and impaired autophagic degradation of protein aggregates might contribute to neuroaxonal dystrophy in this disorder.

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Section: Methodsmentioning

confidence: 99%

Altered distribution of ATG9A and accumulation of axonal aggregates in neurons from a mouse model of AP-4 deficiency syndrome

et al. 2018

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“…Primary hippocampal neurons were prepared from rats and mice as previously described ( Farías et al, 2016 ). In brief, embryonic day 18 (E18) rats or mice were harvested and euthanized by decapitation.…”

Section: Methodsmentioning

confidence: 99%

Synaptic Vesicle Precursors and Lysosomes Are Transported by Different Mechanisms in the Axon of Mammalian Neurons

et al. 2020

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SUMMARY BORC is a multisubunit complex previously shown to promote coupling of mammalian lysosomes and C. elegans synaptic vesicle (SV) precursors (SVPs) to kinesins for anterograde transport of these organelles along microtubule tracks. We attempted to meld these observations into a unified model for axonal transport in mammalian neurons by testing two alternative hypotheses: (1) that SV and lysosomal proteins are co-transported within a single type of “lysosome-related vesicle” and (2) that SVPs and lysosomes are distinct organelles, but both depend on BORC for axonal transport. Analyses of various types of neurons from wild-type rats and mice, as well as from BORC-deficient mice, show that neither hypothesis is correct. We find that SVPs and lysosomes are transported separately, but only lysosomes depend on BORC for axonal transport in these neurons. These findings demonstrate that SVPs and lysosomes are distinct organelles that rely on different machineries for axonal transport in mammalian neurons.

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“…HeLa, COS-7, and HT-1080 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Corning, Corning, NY, USA) supplemented with 10% fetal bovine serum (Corning), 100 IU/mL penicillin and 100 μg/mL streptomycin (Corning), and 2 mM L-glutamine (Corning) in a 37°C incubator with 5% CO 2 and 95% air. Primary cultures of rat hippocampal neurons were prepared at embryonic day 18 and cultured as previously described [69]. Cells were seeded in 24-well plates with glass coverslips or 8-well chambered cover glass (Cellvis, Mountain View, CA, USA).…”

Section: Methodsmentioning

confidence: 99%

Reversible association with motor proteins (RAMP): A streptavidin-based method to manipulate organelle positioning

et al. 2019

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We report the development and characterization of a method, named reversible association with motor proteins (RAMP), for manipulation of organelle positioning within the cytoplasm. RAMP consists of coexpressing in cultured cells (i) an organellar protein fused to the streptavidin-binding peptide (SBP) and (ii) motor, neck, and coiled-coil domains from a plus-end–directed or minus-end–directed kinesin fused to streptavidin. The SBP–streptavidin interaction drives accumulation of organelles at the plus or minus end of microtubules, respectively. Importantly, competition of the streptavidin–SBP interaction by the addition of biotin to the culture medium rapidly dissociates the motor construct from the organelle, allowing restoration of normal patterns of organelle transport and distribution. A distinctive feature of this method is that organelles initially accumulate at either end of the microtubule network in the initial state and are subsequently released from this accumulation, allowing analyses of the movement of a synchronized population of organelles by endogenous motors.

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Imaging the Polarized Sorting of Proteins from the Golgi Complex in Live Neurons

Cited by 20 publications

References 19 publications

Altered distribution of ATG9A and accumulation of axonal aggregates in neurons from a mouse model of AP-4 deficiency syndrome

Altered distribution of ATG9A and accumulation of axonal aggregates in neurons from a mouse model of AP-4 deficiency syndrome

Synaptic Vesicle Precursors and Lysosomes Are Transported by Different Mechanisms in the Axon of Mammalian Neurons

Reversible association with motor proteins (RAMP): A streptavidin-based method to manipulate organelle positioning

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