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Dynein-dynactin complex is essential for dendritic restriction of TM1-containing Drosophila Dscam.

Yang JS, Bai JM, Lee T - PLoS ONE (2008)

Bottom Line: In contrast, compromising dynein/dynactin function did not affect dendritic targeting of two other dendritic markers, Nod and Rdl.Tracing newly synthesized Dscam[TM1] further revealed that compromising dynein/dynactin function did not affect the initial dendritic targeting of Dscam[TM1], but disrupted the maintenance of its restriction to dendrites.The results of this study suggest multiple mechanisms of dendritic protein targeting.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, University of Massachusetts Medical School, Worcester, MA, USA.

ABSTRACT

Background: Many membrane proteins, including Drosophila Dscam, are enriched in dendrites or axons within neurons. However, little is known about how the differential distribution is established and maintained.

Methodology/principal findings: Here we investigated the mechanisms underlying the dendritic targeting of Dscam[TM1]. Through forward genetic mosaic screens and by silencing specific genes via targeted RNAi, we found that several genes, encoding various components of the dynein-dynactin complex, are required for restricting Dscam[TM1] to the mushroom body dendrites. In contrast, compromising dynein/dynactin function did not affect dendritic targeting of two other dendritic markers, Nod and Rdl. Tracing newly synthesized Dscam[TM1] further revealed that compromising dynein/dynactin function did not affect the initial dendritic targeting of Dscam[TM1], but disrupted the maintenance of its restriction to dendrites.

Conclusions/significance: The results of this study suggest multiple mechanisms of dendritic protein targeting. Notably, dynein-dynactin plays a role in excluding dendritic Dscam, but not Rdl, from axons by retrograde transport.

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Mistargeting of dendritic Dscam following depletion of various components of dynein-dynactin complex.(A–G) Distribution of Dscam[TM1]::GFP in the larval MBs where a dynein/dynactin-unrelated gene CG8446 (A) or various components of dynein/dynactin complex (B–G) were silenced by induction of RNAis with GAL4-OK107. Dscam[TM1]::GFP was no longer restricted to the cell bodies and calyx, when dynein/dynactin components were knocked down (B–G, compared to A). Note granular accumulation at the ends of axon lobes in [B] to [D] (arrowheads) versus uniform distribution in [E] to [G] (arrows). Double knockdown (H and I) showed more granular accumulation at the ends of axons than individual knockdowns (E–G) have. (J) Schematic illustration of dynein/dynactin complex. The entire axonal lobes were outlined by dashed lines according to the 1D4 mAb staining (red in A).
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pone-0003504-g003: Mistargeting of dendritic Dscam following depletion of various components of dynein-dynactin complex.(A–G) Distribution of Dscam[TM1]::GFP in the larval MBs where a dynein/dynactin-unrelated gene CG8446 (A) or various components of dynein/dynactin complex (B–G) were silenced by induction of RNAis with GAL4-OK107. Dscam[TM1]::GFP was no longer restricted to the cell bodies and calyx, when dynein/dynactin components were knocked down (B–G, compared to A). Note granular accumulation at the ends of axon lobes in [B] to [D] (arrowheads) versus uniform distribution in [E] to [G] (arrows). Double knockdown (H and I) showed more granular accumulation at the ends of axons than individual knockdowns (E–G) have. (J) Schematic illustration of dynein/dynactin complex. The entire axonal lobes were outlined by dashed lines according to the 1D4 mAb staining (red in A).

Mentions: In order to substantiate the involvement of dynein-dynactin complex, we first confirmed that Lis1, Dmn, and p24 are required for the dendritic restriction of Dscam[TM1]::GFP using reagents independent of our genetic screen. Genes could be effectively silenced in the MBs by RNA interference (RNAi) [27], [40]; and transgenic flies carrying UAS-RNAi against various Drosophila genes, including Lis1, Dmn, and many other components of dynein-dynactin complex, are available in the Vienna Drosophila RNAi Center (VDRC) [41]. Encouragingly, silencing Lis1 or Dmn, as opposed to various control genes (such as CG8446 and CG18247), by targeted RNAi effectively mislocalized transgenic Dscam[TM1]::GFP to MB axon lobes (Figures 3A–3C). These results not only confirmed the roles of Lis1 and Dmn, but also illustrated the utility of RNAi in quickly uncovering more genes in a common pathway. We confirmed the indispensability of p24 in Dscam localization by examining Dscam[TM1]::GFP distribution in MB clones homozygous for a pre-existing loss-of-function allele of p24 (data not shown). Analogous mislocalization phenotypes were obtained when Lis1, Dmn, or p24 were depleted by various means, substantiating their involvement, possibly through the dynein-dynactin complex, in excluding dendritic Dscam from axons.


Dynein-dynactin complex is essential for dendritic restriction of TM1-containing Drosophila Dscam.

Yang JS, Bai JM, Lee T - PLoS ONE (2008)

Mistargeting of dendritic Dscam following depletion of various components of dynein-dynactin complex.(A–G) Distribution of Dscam[TM1]::GFP in the larval MBs where a dynein/dynactin-unrelated gene CG8446 (A) or various components of dynein/dynactin complex (B–G) were silenced by induction of RNAis with GAL4-OK107. Dscam[TM1]::GFP was no longer restricted to the cell bodies and calyx, when dynein/dynactin components were knocked down (B–G, compared to A). Note granular accumulation at the ends of axon lobes in [B] to [D] (arrowheads) versus uniform distribution in [E] to [G] (arrows). Double knockdown (H and I) showed more granular accumulation at the ends of axons than individual knockdowns (E–G) have. (J) Schematic illustration of dynein/dynactin complex. The entire axonal lobes were outlined by dashed lines according to the 1D4 mAb staining (red in A).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2566808&req=5

pone-0003504-g003: Mistargeting of dendritic Dscam following depletion of various components of dynein-dynactin complex.(A–G) Distribution of Dscam[TM1]::GFP in the larval MBs where a dynein/dynactin-unrelated gene CG8446 (A) or various components of dynein/dynactin complex (B–G) were silenced by induction of RNAis with GAL4-OK107. Dscam[TM1]::GFP was no longer restricted to the cell bodies and calyx, when dynein/dynactin components were knocked down (B–G, compared to A). Note granular accumulation at the ends of axon lobes in [B] to [D] (arrowheads) versus uniform distribution in [E] to [G] (arrows). Double knockdown (H and I) showed more granular accumulation at the ends of axons than individual knockdowns (E–G) have. (J) Schematic illustration of dynein/dynactin complex. The entire axonal lobes were outlined by dashed lines according to the 1D4 mAb staining (red in A).
Mentions: In order to substantiate the involvement of dynein-dynactin complex, we first confirmed that Lis1, Dmn, and p24 are required for the dendritic restriction of Dscam[TM1]::GFP using reagents independent of our genetic screen. Genes could be effectively silenced in the MBs by RNA interference (RNAi) [27], [40]; and transgenic flies carrying UAS-RNAi against various Drosophila genes, including Lis1, Dmn, and many other components of dynein-dynactin complex, are available in the Vienna Drosophila RNAi Center (VDRC) [41]. Encouragingly, silencing Lis1 or Dmn, as opposed to various control genes (such as CG8446 and CG18247), by targeted RNAi effectively mislocalized transgenic Dscam[TM1]::GFP to MB axon lobes (Figures 3A–3C). These results not only confirmed the roles of Lis1 and Dmn, but also illustrated the utility of RNAi in quickly uncovering more genes in a common pathway. We confirmed the indispensability of p24 in Dscam localization by examining Dscam[TM1]::GFP distribution in MB clones homozygous for a pre-existing loss-of-function allele of p24 (data not shown). Analogous mislocalization phenotypes were obtained when Lis1, Dmn, or p24 were depleted by various means, substantiating their involvement, possibly through the dynein-dynactin complex, in excluding dendritic Dscam from axons.

Bottom Line: In contrast, compromising dynein/dynactin function did not affect dendritic targeting of two other dendritic markers, Nod and Rdl.Tracing newly synthesized Dscam[TM1] further revealed that compromising dynein/dynactin function did not affect the initial dendritic targeting of Dscam[TM1], but disrupted the maintenance of its restriction to dendrites.The results of this study suggest multiple mechanisms of dendritic protein targeting.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, University of Massachusetts Medical School, Worcester, MA, USA.

ABSTRACT

Background: Many membrane proteins, including Drosophila Dscam, are enriched in dendrites or axons within neurons. However, little is known about how the differential distribution is established and maintained.

Methodology/principal findings: Here we investigated the mechanisms underlying the dendritic targeting of Dscam[TM1]. Through forward genetic mosaic screens and by silencing specific genes via targeted RNAi, we found that several genes, encoding various components of the dynein-dynactin complex, are required for restricting Dscam[TM1] to the mushroom body dendrites. In contrast, compromising dynein/dynactin function did not affect dendritic targeting of two other dendritic markers, Nod and Rdl. Tracing newly synthesized Dscam[TM1] further revealed that compromising dynein/dynactin function did not affect the initial dendritic targeting of Dscam[TM1], but disrupted the maintenance of its restriction to dendrites.

Conclusions/significance: The results of this study suggest multiple mechanisms of dendritic protein targeting. Notably, dynein-dynactin plays a role in excluding dendritic Dscam, but not Rdl, from axons by retrograde transport.

Show MeSH