Vibrator and PI4KIIIα govern neuroblast polarity by anchoring non-muscle myosin II
- Duke-NUS Medical School, Singapore
- Texas A&M University Health Science Center, United States
- National University of Singapore, Singapore
Figures
Figure 1 with 2 supplements
Vibrator regulates homeostasis of larval brain neuroblasts.
(A) A schematic showing the mutation in vib133 allele. (B) Type I MARCM clones of control (FRT82B), vib133, vib1105, UAS-Vib-Venus; vib133 and UAS-Vib-Venus; vib1105 were labeled with Dpn (blue), …
Figure 1—figure supplement 1
Mammalian PITPs rescue vib defects in neuroblast homeostasis.
(A) Western blot of endogenous Vib in larval brain lysate from wild-type (WT), vibj7A3/Df(3R)BSC850, vib133/Df(3R)BSC850 and vib1105/Df(3R)BSC850 hemizygotes. α-tubulin was used as a loading …
Figure 1—figure supplement 2
Loss-of-neuroblast in vib mutants is due to premature differentiation, but not apoptosis.
(A) MARCM clones of control (FRT82B) and vib133 were labeled with Dpn (blue), Cleaved Caspase 3 (red) and CD8::GFP (green). Two different clones were shown for vib133 with one of them containing a …
Figure 2 with 1 supplement
Vib regulates asymmetric cell division of neuroblasts.
(A) Metaphase neuroblasts of wild-type control, vib133/vibj5A6 and vib1105/vibj5A6 were labeled with aPKC, Par-6 or Baz (green) and DNA (blue). (B) Metaphase neuroblasts of control, vib133/vibj5A6 …
Figure 2—figure supplement 1
Vib is required for asymmetric protein localization in neuroblasts.
(A) Metaphase neuroblasts of wild-type control, vib133/vibj5A6 and vib1105/vibj5A6 were labeled with autophospho-aPKC, Gαi or Pins (green) and DNA (blue). (B) Metaphase neuroblasts of wild-type …
Figure 3 with 1 supplement
Vib anchors non-muscle myosin II regulatory light chain Sqh to neuroblast cortex.
(A) Neuroblasts of control, vib133/vibj5A6 and vib1105/vibj5A6 each expressing one copy of Sqh::GFP were labeled with GFP (green), Mira (red) and DNA (blue). (B) Quantification of Sqh::GFP …
Figure 3—figure supplement 1
Sqh localization is disrupted in vib mutant neuroblasts.
(A) Metaphase neuroblasts of control (insc-Gal4/+) and sqh RNAi knockdown (BDSC#33892) under insc-Gal4 driver were labeled with aPKC (green), Mira (red) and DNA (blue). (B) Interphase neuroblasts of …
Figure 4 with 3 supplements
Lipid transfer activity of Vib is important for asymmetric cell division.
(A) Fluorescence intensity measured at 378 nm of PyrPtdCho as a function of protein amount. An increase in intensity represents binding events when the fluorophore is removed from its quenched …
Figure 4—figure supplement 1
Lipid transfer activity of Vib is required for neuroblast homeostasis.
(A) Western blot of Vib protein in larval brain lysate from Vib::Venus, VibT63A::Venus and VibT63E::Venus expression under the elav-Gal4 driver. GAPDH was used as a loading control. (B) Type I and …
Figure 4—figure supplement 2
Lipid binding and transfer activity of Vib is required for the localization of Pros in neuroblasts.
(A) MARCM clones of vib133, UAS-VibT63A::Venus; vib133 and UAS-VibT63E::Venus; vib133 were labeled with Dpn (blue), Ase (red) and CD8 (green). Clones were marked by CD8::GFP and indicated by white …
Figure 4—figure supplement 3
Lipid binding and transfer activity of Vib is required for the localization of Sqh in neuroblasts.
(A) Neuroblasts of wild-type controls, insc-Gal4 >VibT63A, insc-Gal4 >VibT63E, vib133/vibj5A6, insc-Gal4 >VibT63A in vib133/vibj5A6 and insc-Gal4 >VibT63E in vib133/vibj5A6 were labeled with Mira …
Figure 5 with 1 supplement
Sqh binds to phosphoinositide PI(4)P and localizes it to cell cortex in neuroblasts.
(A) Control (empty Myc vector) and Myc-Sqh lysates from S2 cells were used for lipid-binding assay with lipid strips. Lipid strips were blotted with Myc antibody. 1% of lysate input used for …
Figure 5—figure supplement 1
PI(4)P is localized to cell cortex in neuroblasts.
(A) A schematic diagram showing phosphoinositides metabolism. (B) Neuroblasts of PI(4)P-GFP (insc >2xOsh2PH::GFP, PI(4)P reporter) were labeled with GFP (green), Mira (red) and DNA (blue). Scale …
Figure 6 with 1 supplement
Vib interacts with Sqh in larval brain neuroblasts.
(A) In vivo BiFC assay between Vib and Sqh. NYFP-Myc-Vib and CYFP-HA-Sqh were co-expressed in neuroblasts by insc-Gal4, stained with HA (red), Myc (red) and DAPI (grey), and detected for YFP …
Figure 6—figure supplement 1
Vib interacts with Sqh in Bimolecular fluorescence complementation.
S2 cells that were triple transfected with actin-Gal4, UAS-NYFP-Myc-Vib and UAS-CFYP-HA-Sqh or various control combinations were stained with Myc (blue), HA (red) and detected for YFP fluorescence …
Figure 7 with 2 supplements
Loss of PI4KIIIα perturbs neuroblast homeostasis and asymmetric cell division of neuroblasts.
(A) MARCM clones of control (FRT19A) and PI4KIIIαC were labeled with Dpn (blue), Ase (red) and CD8 (green). Arrows indicate neuroblasts. Clones were marked by CD8::GFP and indicated by white dotted …
Figure 7—figure supplement 1
PI4KIIIα, but not PI4KIIIβ or PI4KIIα regulates neuroblast asymmetric division and homeostasis.
(A) Type I and type II MARCM clones of control and PI4KIIΔ were labeled with Dpn (blue), Ase (red) and CD8 (green). Arrows indicate neuroblasts. Clones were marked by CD8::GFP and indicated by white …
Figure 7—figure supplement 2
PI4KIIIα does not regulate Rok localization in neuroblasts.
Neuroblasts expressing Ubi-Rok::GFP were treated with mock (DMSO) or 2.5 μM PAO in DMSO and were labeled with GFP (green), Mira (green) and DNA (blue). Both metaphase and telophase neuroblasts were …
Author response image 1
Tables
Table 1
List of primers used
https://doi.org/10.7554/eLife.33555.021| Primer name | Primer sequence (5'−3') | Purpose |
|---|---|---|
| Vib pENTR F | CACC ATG CAG ATC AAA GAA TTC CGT GTG | Generate Vib pENTR |
| Vib pENTR R | TTA ATC GGC ATC CGC GCG C | |
| Vib pENTR CT R | ATC GGC ATC CGC GCG CAT | Generate Vib pENTR without stop codon |
| Vib T63A F | TAC AAT TCC GGT CAA TAT GCC TAT AAG | Generate T63A mutation |
| Vib T63A R | CTT ATA GGC ATA TTG ACC GGA ATT GTA | |
| Vib T63E F | TAC AAT TCC GGT CAA TAT GAG TAT AAG | Generate T63E mutation |
| Vib T63E R | CTT ATA CTC ATA TTG ACC GGA ATT GTA | |
| Sqh pENTR F | CACC ATG TCA TCC CGT AAG ACC GC | Generate Sqh pENTR |
| Sqh pENTR R | TTA CTG CTC ATC CTT GTC CTT GG | |
| PITPα pENTR F | CACC ATG GTG CTG CTC AAG GAA TA | Generate PITPα pENTR |
| PITPα pENTR R | GTC ATC TGC TGT CAT TCC TTT CA | |
| PITPβ pENTR F | CACC ATG GTG CTG ATT AAG GAA TTC CG | Generate PITPβ pENTR |
| PITPβ pENTR R | GGC ATC AGC AGC CGA CGT GC | |
| Modifier pD221 F | GGGG ACAAGTTTGTACAAAAAAGCAGGCTTC GGTACCGCTGAAACGAAGTTAAACTTTGAGGTGTACGG GTAAGTATTAGAAAGCAGGACTAAACG | Modify pDONR221 to remove RfB cassette from BiFC constructs |
| Modifier pD221 R | GGGG ACCACTTTGTACAAGAAAGCTGGGTC AAGCTTCTATTCAATGGTCCGGCGGCCGACGACATGAGG ATATGGTCGTTTAGTCCTGCTTTCTAA |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.33555.022
