Calcium/Calmodulin-Dependent Protein Kinase II Alters Structural Plasticity and Cytoskeletal Dynamics inDrosophila

Abstract

Drosophila dendritic arborization (da) neurons contain subclasses of neurons with distinct dendritic morphologies. We investigated calcium/calmodulin-dependent protein kinase II (CaMKII) regulation of dendritic structure and dynamics in vivo using optically transparent Drosophila larvae. CaMKII increases the dynamic nature and formation of dendritic filopodia throughout larval development but only affects neurons that normally contain dendritic filopodia. In parallel, we examined the effects of Rac1 activity on dendritic structure to explore signaling specificity. In contrast to CaMKII activity, Rac1 does not alter filopodia stability but instead causes de novo filopodia formation on all da neurons. Although both mediators increase cytoskeletal turnover, measured by fluorescence recovery after photobleaching experiments, only CaMKII increases the dynamic nature of dendritic filopodia. CaMKII signaling thus appears to use mechanisms and machinery distinct from Rac1 signaling. This study illustrates a molecular means of uncoupling cytoskeletal regulation from morphological regulation. Our results suggest that Drosophila dendritic filopodia may share some cytoskeletal regulatory mechanisms with mammalian dendritic filopodia. Furthermore, general dendrite cytoskeletal compartmentalization is conserved in multipolar neurons.

The RhoGEF Trio Functions in Sculpting Class Specific Dendrite Morphogenesis in Drosophila Sensory Neurons

“…6C ). These data suggest that the Trio-GEF1 mediated activation of Rac1 is likely responsible for the increased branching in filopodia and is consistent with previous reports documenting the same phenotype following Rac1 overexpression in class III neurons [21] . Quantitative analyses confirm that Trio-GEF1 upregulation contributes to a significant increase in dendritic branching both proximal and distal to the cell body ( Fig.…”

The RhoGEF Trio Functions in Sculpting Class Specific Dendrite Morphogenesis in Drosophila Sensory Neurons

“…6C ). These data suggest that the Trio-GEF1 mediated activation of Rac1 is likely responsible for the increased branching in filopodia and is consistent with previous reports documenting the same phenotype following Rac1 overexpression in class III neurons [21] . Quantitative analyses confirm that Trio-GEF1 upregulation contributes to a significant increase in dendritic branching both proximal and distal to the cell body ( Fig.…”

Unc-51/ATG1 Controls Axonal and Dendritic Development via Kinesin-Mediated Vesicle Transport in the Drosophila Brain

“…In wild-type MBs, Nod::β-Gal was detected in cell bodies and specifically targeted to the calyx with virtually no signals in the lobes ( Fig. S9G–I ), confirming previous observations [9] , [51] , [52] . Although unc-51 mutant MBs exhibited accumulations in the cell bodies, Nod::β-Gal was specifically targeted to the calyx in all the samples examined ( n = 14) ( Fig.…”

Dynamic recruitment of the curvature-sensitive protein ArhGAP44 to nanoscale membrane deformations limits exploratory filopodia initiation in neurons

“…We propose that ArhGAP44 is limiting Rac-dependent formation of actin patches, which provide the structural integrity required for filopodia to protrude outwards ( Figure 6 ). This is consistent with previous reports in neurons, showing that dynamic rearrangements within actin patches relies on Rac activity ( Andersen et al, 2005 ; Spillane et al, 2012 ), and that elevated Rac levels increase filopodia dynamics ( Luo et al, 1996 ; Nakayama et al, 2000 ; Zhang and Macara, 2006 ; Cheadle and Biederer, 2012 ). In support of this notion, we find Rac1 localized at actin patches ( Figure 6—figure supplement 1A ) and show that artificial decrease of ArhGAP44 concentration at actin patches either by reducing overall ArhGAP44 levels by knockdown ( Figure 1F ) or by preventing ArhGAP44-recruitment to actin patches via inhibition of acto-myosin contraction ( Figure 6—figure supplement 1B , see also [ Ryu et al, 2006 ; Hodges et al, 2011 ]) both increased the number of exploratory filopodia.…”

The RhoGEF Trio Functions in Sculpting Class Specific Dendrite Morphogenesis in Drosophila Sensory Neurons

“…6C ). These data suggest that the Trio-GEF1 mediated activation of Rac1 is likely responsible for the increased branching in filopodia and is consistent with previous reports documenting the same phenotype following Rac1 overexpression in class III neurons [21] . Quantitative analyses confirm that Trio-GEF1 upregulation contributes to a significant increase in dendritic branching both proximal and distal to the cell body ( Fig.…”

Unc-51/ATG1 Controls Axonal and Dendritic Development via Kinesin-Mediated Vesicle Transport in the Drosophila Brain

“…In wild-type MBs, Nod::β-Gal was detected in cell bodies and specifically targeted to the calyx with virtually no signals in the lobes ( Fig. S9G–I ), confirming previous observations [9] , [51] , [52] . Although unc-51 mutant MBs exhibited accumulations in the cell bodies, Nod::β-Gal was specifically targeted to the calyx in all the samples examined ( n = 14) ( Fig.…”

Dynamic recruitment of the curvature-sensitive protein ArhGAP44 to nanoscale membrane deformations limits exploratory filopodia initiation in neurons

“…We propose that ArhGAP44 is limiting Rac-dependent formation of actin patches, which provide the structural integrity required for filopodia to protrude outwards ( Figure 6 ). This is consistent with previous reports in neurons, showing that dynamic rearrangements within actin patches relies on Rac activity ( Andersen et al, 2005 ; Spillane et al, 2012 ), and that elevated Rac levels increase filopodia dynamics ( Luo et al, 1996 ; Nakayama et al, 2000 ; Zhang and Macara, 2006 ; Cheadle and Biederer, 2012 ). In support of this notion, we find Rac1 localized at actin patches ( Figure 6—figure supplement 1A ) and show that artificial decrease of ArhGAP44 concentration at actin patches either by reducing overall ArhGAP44 levels by knockdown ( Figure 1F ) or by preventing ArhGAP44-recruitment to actin patches via inhibition of acto-myosin contraction ( Figure 6—figure supplement 1B , see also [ Ryu et al, 2006 ; Hodges et al, 2011 ]) both increased the number of exploratory filopodia.…”

The RhoGEF Trio Functions in Sculpting Class Specific Dendrite Morphogenesis in Drosophila Sensory Neurons

“…6C ). These data suggest that the Trio-GEF1 mediated activation of Rac1 is likely responsible for the increased branching in filopodia and is consistent with previous reports documenting the same phenotype following Rac1 overexpression in class III neurons [21] . Quantitative analyses confirm that Trio-GEF1 upregulation contributes to a significant increase in dendritic branching both proximal and distal to the cell body ( Fig.…”

Unc-51/ATG1 Controls Axonal and Dendritic Development via Kinesin-Mediated Vesicle Transport in the Drosophila Brain

“…In wild-type MBs, Nod::β-Gal was detected in cell bodies and specifically targeted to the calyx with virtually no signals in the lobes ( Fig. S9G–I ), confirming previous observations [9] , [51] , [52] . Although unc-51 mutant MBs exhibited accumulations in the cell bodies, Nod::β-Gal was specifically targeted to the calyx in all the samples examined ( n = 14) ( Fig.…”

Dynamic recruitment of the curvature-sensitive protein ArhGAP44 to nanoscale membrane deformations limits exploratory filopodia initiation in neurons

“…We propose that ArhGAP44 is limiting Rac-dependent formation of actin patches, which provide the structural integrity required for filopodia to protrude outwards ( Figure 6 ). This is consistent with previous reports in neurons, showing that dynamic rearrangements within actin patches relies on Rac activity ( Andersen et al, 2005 ; Spillane et al, 2012 ), and that elevated Rac levels increase filopodia dynamics ( Luo et al, 1996 ; Nakayama et al, 2000 ; Zhang and Macara, 2006 ; Cheadle and Biederer, 2012 ). In support of this notion, we find Rac1 localized at actin patches ( Figure 6—figure supplement 1A ) and show that artificial decrease of ArhGAP44 concentration at actin patches either by reducing overall ArhGAP44 levels by knockdown ( Figure 1F ) or by preventing ArhGAP44-recruitment to actin patches via inhibition of acto-myosin contraction ( Figure 6—figure supplement 1B , see also [ Ryu et al, 2006 ; Hodges et al, 2011 ]) both increased the number of exploratory filopodia.…”