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The vesicle protein SAM-4 regulates the processivity of synaptic vesicle transport.

Zheng Q, Ahlawat S, Schaefer A, Mahoney T, Koushika SP, Nonet ML - PLoS Genet. (2014)

Bottom Line: Processivity, but not velocity, of SV transport was reduced in sam-4 mutants. sam-4 displayed strong genetic interactions with mutations in the cargo binding but not the motor domain of unc-104.Gain-of-function mutations in the unc-104 motor domain, identified in this study, suppress the sam-4 defects by increasing processivity of the SV transport.Our data support a model in which the SV protein SAM-4 regulates the processivity of SV transport.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Neurobiology, Washington University Medical School, St. Louis, Missouri, United States of America.

ABSTRACT
Axonal transport of synaptic vesicles (SVs) is a KIF1A/UNC-104 mediated process critical for synapse development and maintenance yet little is known of how SV transport is regulated. Using C. elegans as an in vivo model, we identified SAM-4 as a novel conserved vesicular component regulating SV transport. Processivity, but not velocity, of SV transport was reduced in sam-4 mutants. sam-4 displayed strong genetic interactions with mutations in the cargo binding but not the motor domain of unc-104. Gain-of-function mutations in the unc-104 motor domain, identified in this study, suppress the sam-4 defects by increasing processivity of the SV transport. Genetic analyses suggest that SAM-4, SYD-2/liprin-α and the KIF1A/UNC-104 motor function in the same pathway to regulate SV transport. Our data support a model in which the SV protein SAM-4 regulates the processivity of SV transport.

No MeSH data available.


Related in: MedlinePlus

js1288 and js1289 are gain-of-function mutations in the motor domain of the unc-104 gene.(A–D) Effects of unc-104 overexpression (OE, jsIs1111) on TagRFP-RAB-3 (jsIs1263) accumulation in PLM synaptic varicosities of wild type (A and C) and sam-4 (js415) (B and D). (E–G″) Distribution of the SV marker GFP-RAB-3 in PLM neurons. Shown are representative images of the GFP-RAB-3 (jsIs821) signal observed in the distal end of PLM neurites (E–G), the PLM synaptic varicosities (E′–G′), and the PLM soma (E″–G″). Arrow: PLM soma; arrowhead: PLM synaptic varicosity; asterisk: vulva. Scale bar: 20 µm. (H–J) Quantification of GFP- RAB-3 fluorescence intensities in the distal end of the neurite (30 µm) (H), the synaptic varicosities (I), and the soma (J). Note that, due to differences between imaging conditions for individual PLM anatomic regions, arbitrary units and fluorescence intensity between regions are not comparable. (K) Quantification of TagRFP-RAB-3 fluorescence intensity in PLM synaptic varicosities in different genetic backgrounds. *, P<0.01 relative to wild type; **, P<0.001 relative to wild type; ΔΔ, P<0.001 relative to sam-4(js415).
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pgen-1004644-g007: js1288 and js1289 are gain-of-function mutations in the motor domain of the unc-104 gene.(A–D) Effects of unc-104 overexpression (OE, jsIs1111) on TagRFP-RAB-3 (jsIs1263) accumulation in PLM synaptic varicosities of wild type (A and C) and sam-4 (js415) (B and D). (E–G″) Distribution of the SV marker GFP-RAB-3 in PLM neurons. Shown are representative images of the GFP-RAB-3 (jsIs821) signal observed in the distal end of PLM neurites (E–G), the PLM synaptic varicosities (E′–G′), and the PLM soma (E″–G″). Arrow: PLM soma; arrowhead: PLM synaptic varicosity; asterisk: vulva. Scale bar: 20 µm. (H–J) Quantification of GFP- RAB-3 fluorescence intensities in the distal end of the neurite (30 µm) (H), the synaptic varicosities (I), and the soma (J). Note that, due to differences between imaging conditions for individual PLM anatomic regions, arbitrary units and fluorescence intensity between regions are not comparable. (K) Quantification of TagRFP-RAB-3 fluorescence intensity in PLM synaptic varicosities in different genetic backgrounds. *, P<0.01 relative to wild type; **, P<0.001 relative to wild type; ΔΔ, P<0.001 relative to sam-4(js415).

Mentions: To further understand how SAM-4 activity regulates SV trafficking, we conducted a genetic screen for sam-4 suppressors. Using ENU induced mutagenesis, we screened mutated progeny of sam-4(js415); jsIs821 for animals with increased GFP-RAB-3 signal in PLM synaptic varicosities (Figure 6A–6D) and isolated two suppressors from roughly 100,000 genomes screened. Combining traditional genetic mapping and whole genome sequencing strategies, we identified both mutations as novel unc-104 alleles (see Materials and Methods for details). Interestingly, we found that the alleles introduce missense mutations in the UNC-104/KIF1A motor domain: S211A (js1288) and D177A (js1289), both of which are conserved in mammalian molecular motor proteins (Figure S9). Further genetic tests showed that both alleles are semi-dominant in suppressing sam-4 defects. Additionally, we found that over-expression of wild type unc-104 (unc-104(+)) in PLM neurons suppresses sam-4 defects (Figure 7A–7D and 7K), but over-expression of sam-4 does not suppress unc-104 defects (Figure S7). Similar suppression analysis using syd-2 mutants by these unc-104(gf) mutations also revealed suppression by unc-104(gf) alleles (Figure 6). Taken together, these data argue that both js1288 and js1289 are gain-of-function alleles of unc-104, and unc-104 is epistatic to sam-4 and syd-2.


The vesicle protein SAM-4 regulates the processivity of synaptic vesicle transport.

Zheng Q, Ahlawat S, Schaefer A, Mahoney T, Koushika SP, Nonet ML - PLoS Genet. (2014)

js1288 and js1289 are gain-of-function mutations in the motor domain of the unc-104 gene.(A–D) Effects of unc-104 overexpression (OE, jsIs1111) on TagRFP-RAB-3 (jsIs1263) accumulation in PLM synaptic varicosities of wild type (A and C) and sam-4 (js415) (B and D). (E–G″) Distribution of the SV marker GFP-RAB-3 in PLM neurons. Shown are representative images of the GFP-RAB-3 (jsIs821) signal observed in the distal end of PLM neurites (E–G), the PLM synaptic varicosities (E′–G′), and the PLM soma (E″–G″). Arrow: PLM soma; arrowhead: PLM synaptic varicosity; asterisk: vulva. Scale bar: 20 µm. (H–J) Quantification of GFP- RAB-3 fluorescence intensities in the distal end of the neurite (30 µm) (H), the synaptic varicosities (I), and the soma (J). Note that, due to differences between imaging conditions for individual PLM anatomic regions, arbitrary units and fluorescence intensity between regions are not comparable. (K) Quantification of TagRFP-RAB-3 fluorescence intensity in PLM synaptic varicosities in different genetic backgrounds. *, P<0.01 relative to wild type; **, P<0.001 relative to wild type; ΔΔ, P<0.001 relative to sam-4(js415).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4199485&req=5

pgen-1004644-g007: js1288 and js1289 are gain-of-function mutations in the motor domain of the unc-104 gene.(A–D) Effects of unc-104 overexpression (OE, jsIs1111) on TagRFP-RAB-3 (jsIs1263) accumulation in PLM synaptic varicosities of wild type (A and C) and sam-4 (js415) (B and D). (E–G″) Distribution of the SV marker GFP-RAB-3 in PLM neurons. Shown are representative images of the GFP-RAB-3 (jsIs821) signal observed in the distal end of PLM neurites (E–G), the PLM synaptic varicosities (E′–G′), and the PLM soma (E″–G″). Arrow: PLM soma; arrowhead: PLM synaptic varicosity; asterisk: vulva. Scale bar: 20 µm. (H–J) Quantification of GFP- RAB-3 fluorescence intensities in the distal end of the neurite (30 µm) (H), the synaptic varicosities (I), and the soma (J). Note that, due to differences between imaging conditions for individual PLM anatomic regions, arbitrary units and fluorescence intensity between regions are not comparable. (K) Quantification of TagRFP-RAB-3 fluorescence intensity in PLM synaptic varicosities in different genetic backgrounds. *, P<0.01 relative to wild type; **, P<0.001 relative to wild type; ΔΔ, P<0.001 relative to sam-4(js415).
Mentions: To further understand how SAM-4 activity regulates SV trafficking, we conducted a genetic screen for sam-4 suppressors. Using ENU induced mutagenesis, we screened mutated progeny of sam-4(js415); jsIs821 for animals with increased GFP-RAB-3 signal in PLM synaptic varicosities (Figure 6A–6D) and isolated two suppressors from roughly 100,000 genomes screened. Combining traditional genetic mapping and whole genome sequencing strategies, we identified both mutations as novel unc-104 alleles (see Materials and Methods for details). Interestingly, we found that the alleles introduce missense mutations in the UNC-104/KIF1A motor domain: S211A (js1288) and D177A (js1289), both of which are conserved in mammalian molecular motor proteins (Figure S9). Further genetic tests showed that both alleles are semi-dominant in suppressing sam-4 defects. Additionally, we found that over-expression of wild type unc-104 (unc-104(+)) in PLM neurons suppresses sam-4 defects (Figure 7A–7D and 7K), but over-expression of sam-4 does not suppress unc-104 defects (Figure S7). Similar suppression analysis using syd-2 mutants by these unc-104(gf) mutations also revealed suppression by unc-104(gf) alleles (Figure 6). Taken together, these data argue that both js1288 and js1289 are gain-of-function alleles of unc-104, and unc-104 is epistatic to sam-4 and syd-2.

Bottom Line: Processivity, but not velocity, of SV transport was reduced in sam-4 mutants. sam-4 displayed strong genetic interactions with mutations in the cargo binding but not the motor domain of unc-104.Gain-of-function mutations in the unc-104 motor domain, identified in this study, suppress the sam-4 defects by increasing processivity of the SV transport.Our data support a model in which the SV protein SAM-4 regulates the processivity of SV transport.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Neurobiology, Washington University Medical School, St. Louis, Missouri, United States of America.

ABSTRACT
Axonal transport of synaptic vesicles (SVs) is a KIF1A/UNC-104 mediated process critical for synapse development and maintenance yet little is known of how SV transport is regulated. Using C. elegans as an in vivo model, we identified SAM-4 as a novel conserved vesicular component regulating SV transport. Processivity, but not velocity, of SV transport was reduced in sam-4 mutants. sam-4 displayed strong genetic interactions with mutations in the cargo binding but not the motor domain of unc-104. Gain-of-function mutations in the unc-104 motor domain, identified in this study, suppress the sam-4 defects by increasing processivity of the SV transport. Genetic analyses suggest that SAM-4, SYD-2/liprin-α and the KIF1A/UNC-104 motor function in the same pathway to regulate SV transport. Our data support a model in which the SV protein SAM-4 regulates the processivity of SV transport.

No MeSH data available.


Related in: MedlinePlus