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Results: 9

1.
Figure 7

Figure 7. Downregulation of PTEN affects bouton number and bouton volume. From: A critical step for postsynaptic F-actin organization: Regulation of Baz/Par-3 localization by aPKC and PTEN.

(A–C) Quantification of (A, C) bouton number in the indicated genoptyes (n represents number of arbors), and (B) normalized bouton volume (n represents number of boutons).

Preethi Ramachandran, et al. Dev Neurobiol. ;69(9):583-602.
2.
Figure 5

Figure 5. PTEN is localized at the postsynaptic F-actin-rich area. From: A critical step for postsynaptic F-actin organization: Regulation of Baz/Par-3 localization by aPKC and PTEN.

(A) A model of Baz trafficking at the NMJ. See text for details.
(B–C) Quantification of (B) normalized postsynaptic PTEN fluorescence intensity (black bars) and normalized volume (white bars) in the indicated genotypes (n represents number of boutons) and (C) normalized cortical PTEN intensity in the indicated genotypes (n represents number of muscles).
(D–E) Third instar NMJs at muscles 6 or 7 in preparation double stained with anti-PTEN (green) and anti-HRP (red) in (D) wild type and (E) pten/+; PTEN-RNAi-post.
(F) Third instar NMJ from wild type triple labeled with rhodamine-phalloidin (red), anti-PTEN (green) and anti-HRP (blue).
Calibration bar is 14 μm for DF.

Preethi Ramachandran, et al. Dev Neurobiol. ;69(9):583-602.
3.
Figure 9

Figure 9. Proposed model for the regulation of the synaptic cytoskeleton by the aPKC-Baz-Par-6 complex at the larval NMJ. From: A critical step for postsynaptic F-actin organization: Regulation of Baz/Par-3 localization by aPKC and PTEN.

(A) At the presynaptic compartment, aPKC regulates microtubule stability by facilitating an interaction between the MAP1B-related Futsch and bundled microtubules (see (Ruiz-Canada et al., 2004)). At the postsynaptic compartment, aPKC regulates both F-actin and microtubules (Ruiz-Canada et al., 2004). Here we propose that the regulation of the F-actin cytoskeleton is carried out through the opposing functions of aPKC and PTEN by modulating the phosphorylation state of Baz (see text for details).
In (B) the synaptic bouton has been subdivided into 4 regions, each representing a different genotype as indicated, and the consequence of each genotype on the F-actin-rich postsynaptic region (see text for details).

Preethi Ramachandran, et al. Dev Neurobiol. ;69(9):583-602.
4.
Figure 8

Figure 8. Downregulation of aPKC, PTEN and Baz have similar effects on synaptic transmission and glutamate receptor localization. From: A critical step for postsynaptic F-actin organization: Regulation of Baz/Par-3 localization by aPKC and PTEN.

(A–D) Third instar larval NMJs labeled with HRP (blue) and GluRIIA (red) in (A) wild type, (B) aPKCs-RNAi-post, (C) baz/+; Baz-RNAi-post, and (D) pten/+; PTEN-RNAi-post.
(E, F) Electrophysiological traces of (E) EJPs and (F) mEJPs in the indicated genotypes.
(G, H, I, J) Quantification of (G) GluRIIA cluster volume (black bars), mean intensity (blue bars), and total intensity (red bars) (n represents number of clusters), (H) mEJP amplitude, (I) mini EJP frequency, and (K) EJP amplitude in the indicated genotypes. In (HJ) n=6 animals for all genotypes.
Calibration bar is 7 μm for A–D.

Preethi Ramachandran, et al. Dev Neurobiol. ;69(9):583-602.
5.
Figure 3

Figure 3. Baz is enriched in the F-actin region and reduction in Baz levels phenocopy aPKC downregulation at the NMJ. From: A critical step for postsynaptic F-actin organization: Regulation of Baz/Par-3 localization by aPKC and PTEN.

Single confocal slices of third instar larval NMJs,
(A–B) Triple stained with anti-HRP (blue), anti-Baz (green) and rhodamine-phalloidin (red) in (A) wild type or (B) aPKC-RNAi-Post (muscle 12).
(C–D) Double stained with anti-HRP (blue) and anti-Baz (green) in (C) wild type and (D) baz/+; Baz RNAi-post (muscle 6 or 7).
(E–H) Double stained with anti-HRP (blue) and (E–F) rhodamine-phalloidin (red), or (G–H) anti-α-spectrin (green) in (E,G) wild type (F, H) baz/+; Baz RNAi-post (muscles 12/13 for F-actin and mudcles 6/7 for spectrin)
(I–M) Quantification of (I) normalized postsynaptic Baz intensity (“n” represents number of boutons”, (J) normalized postsynaptic F-actin (black bars, muscles 12/13), and spectrin (white bars, muscles 6/7) intensity (“n” represents number of boutons), (K) normalized postsynaptic volume (n is the same as in J), (L) bouton number (n represents number of arbors), and (M) bouton volume (n represents number of boutons) in the indicated genotypes.
Calibration bar is 15 μm for A, B, and 10 μm for CH.

Preethi Ramachandran, et al. Dev Neurobiol. ;69(9):583-602.
6.
Figure 6

Figure 6. PTEN induces dephosphorylation of Bazooka and affects the postsynaptic actin spectrin area. From: A critical step for postsynaptic F-actin organization: Regulation of Baz/Par-3 localization by aPKC and PTEN.

(A–B) Third instar NMJs at muscles 6 or 7 double stained with anti-HRP (red) and anti-Baz (green) in (A) wild type, (B) PTEN-RNAi-post. Note the reduction in postsynaptic Baz in PTEN-RNAi-post compared to the wild type as indicated by arrows in A3, B3.
(C–F)Third instar NMJs stained with anti-HRP (red) and either (C,D) anti-α-spectrin (green), or (E,F) anti-phospho-Baz (green) in (C,E) wild type and (D,F) PTEN-RNAi-Post.
(G–H) Third instar NMJs stained with anti-HRP (red) and anti-PTEN (green) in (G) wild type and (H) baz/+; Baz-RNAi-post.
(I, J) Quantification of (I) normalized volume and (J) normalized intensity of α-spectrin (black bars; muscles 6 or 7) and F-actin (white bars, muscle 12) in the indicated genotypes. n represents number of boutons, and n is the same for I and J.
(K) Quantification of normalized postsynaptic Baz volume (black bars) and normalized postsynaptic Baz fluorescence intensity (white bars) in the indicated genotypes. n represents number of boutons.
(L) Quantification of normalized phospho-Baz fluorescence intensity in wild type, PTEN RNAi-post and pten/+; PTEN RNAi-post. n represents number of muscles.
(M) Western blot of S2 cell extracts from cells transfected with the genes shown above the blots and probed sequentially with the antibodies indicated at the left, showing the dephosphorylation of Baz by PTEN.
Calibration bar is 7μm for A1–2, B1–2, CH and 3.5 μm for A3, B3.

Preethi Ramachandran, et al. Dev Neurobiol. ;69(9):583-602.
7.
Figure 2

Figure 2. Localization of Spectrin and F-actin at NMJs of wild type, aPKC RNAi-post, and β-specem6/+ mutants. From: A critical step for postsynaptic F-actin organization: Regulation of Baz/Par-3 localization by aPKC and PTEN.

(A, B,C) Single confocal slices of third instar NMJ branches at muscles12/13 in preparations, triple stained with anti-HRP (blue), anti-α-spectrin (green), and rhodamine-phalloidin (red) in (A), wild type, (B) larvae expressing aPKC-RNAi in muscles, (C) β-specem6/+ mutants.
(D, E, F) are high magnification views of synaptic boutons in the above genotypes stained with anti-HRP (blue) and rhodamine-phalloidin (red) showing the distribution of F-actin around the presynaptic compartment labeled by anti-HRP.
(G) Extended view of an NMJ at muscle 12/13 in a β-specem6/+ heterozygote larval body wall muscle preparation stained with anti-HRP (blue) and rhodamine-phalloidin (red) showing the organization of F-actin into wisps/spikes. Arrows point to the F-actin-rich postsynaptic area.
(H, I) Quantification of the (H) normalized volume and (I) normalized fluorescence intensityof postsynaptic α-spectrin (white bars) and F-actin (black bars) in the indicated genotypes. Data was normalized to wild type controls. “n” represents number of boutons.
Calibration bar is 7 μm for A, B, C, 2 μm for D, E, F, and 10 μm for G.

Preethi Ramachandran, et al. Dev Neurobiol. ;69(9):583-602.
8.
Figure 4

Figure 4. Baz is phosphorylated by aPKC in muscles and is absent from the postsynaptic F-actin-rich area. From: A critical step for postsynaptic F-actin organization: Regulation of Baz/Par-3 localization by aPKC and PTEN.

(A–E) Third instar muscle regions and NMJs at muscles 6 or 7 in preparations labeled with anti-phospho Baz (green) and anti-HRP (blue) in (A, D) wild type, (B) PKM-post, (C) baz/+; Baz-RNAi-post and (E) aPKC-RNAi-post. Arrow in D points to the postsynaptic F-actin rich area outlined by a white line, which is devoid of phospho-Baz.
(F) Quantification of the volume and intensity of phospho-Baz puncta in muscles of wild type, aPKC-RNAi-post, and PKM-post. Data was normalized to wild type values (n represents number of muscles).
(G, I) Confocal image of a muscle region in a preparation (G) sequentially labeled with antibodies to P-Baz (green) and Baz (red) showing that each P-Baz puncta colocalizes to a Baz puncta (arrows), and that only a subset of Baz puncta contains phosphorylated P-Baz (arrowheads point to Baz puncta devoid of P-Baz immunoreactivity), and (I) treated similar to G, but in an experiment were the anti-Baz antibody was omitted to ensure that the first secondary antibody (FITC 2nd) saturated all anti-P-Baz sites, which is observed in I2 by the lack of signal upon incubation with the TxR-2nd antibody.
(H, J) Western blots of S2 cell extracts transfected with the constructs indicated above the blots and probed sequentially with the antibodies indicated at the left, showing (H) the phosphorylation of Baz by PKM and (J) the lambda phosphatase assay. Untrans=untransfected cells.
Calibration bar is 14 μm for A, B, C, E, 5 μm for D, and 2 μm for G.

Preethi Ramachandran, et al. Dev Neurobiol. ;69(9):583-602.
9.
Figure 1

Figure 1. Arrangement of postsynaptic F-actin and the SSR in relationship to the contractile apparatus. From: A critical step for postsynaptic F-actin organization: Regulation of Baz/Par-3 localization by aPKC and PTEN.

(A) Diagram of the NMJ showing the relative positions of post-synaptic F-actin/spectrin, aPKC, microtubules, the SSR and the contractile apparatus. N= nucleus.
(B) 3-D rendered confocal images of a wild type arbor at muscle 12 labeled with anti-HRP (blue) and rhodamine-phalloidin (red) in (left panel; also see Suppl. Movie 1) a X-Y-Z 3-D isosurface rendering showing a single NMJ branch containing synaptic boutons (blue) surrounded by postsynaptic F-actin-rich domain (red) sitting above the contractile apparatus (red) of the muscle. In the right panel, the same image is now shown in a YZ plane, demonstrating that postsynaptic F-actin (red) surrounding synaptic boutons (blue) appears separate from the F-actin at the contractile apparatus (distance marked by a two-way arrow). Arrow and arrowhead point to the postsynaptic actin.
(C) Transmission electron micrograph through an NMJ branch at muscle 12 (m12) showing several boutons (Ib, Is, III) and a region of the contractile apparatus near the NMJ branch-point. Red two-way arrows show the distance between the SSR and the myofibrils of the contractile apparatus in these hypercontractile muscles.
(D–F) High magnification views of myofibril regions showing (D) perforated Z-bands, (E) the myosin-actin lattice and the sarcoplasmic reticulum (sr), and (F) the dyads composed of the t-tubule (tt) and sr compartments.
(G) View of the nerve as it runs from muscle 13 (m13) to muscle 12 and the NMJ branch-point at muscle 12. At this region the distance between the SSR and muscle contractile apparatus (two-way arrow) is larger. N= nucleus; Z= perforated Z band; mi =mitochondria; ssr= subsynaptic reticulum.
Calibration bar is 3.5 μm in B, 0.4 μm in C, G and 80 nm in D–F.

Preethi Ramachandran, et al. Dev Neurobiol. ;69(9):583-602.

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