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PLoS Genet
2008 Feb 01;42:e36. doi: 10.1371/journal.pgen.0040036.
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Motility screen identifies Drosophila IGF-II mRNA-binding protein--zipcode-binding protein acting in oogenesis and synaptogenesis.
Boylan KL
,
Mische S
,
Li M
,
Marqués G
,
Morin X
,
Chia W
,
Hays TS
.
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The localization of specific mRNAs can establish local protein gradients that generate and control the development of cellular asymmetries. While all evidence underscores the importance of the cytoskeleton in the transport and localization of RNAs, we have limited knowledge of how these events are regulated. Using a visual screen for motile proteins in a collection of GFP protein trap lines, we identified the Drosophila IGF-II mRNA-binding protein (Imp), an ortholog of Xenopus Vg1 RNA binding protein and chicken zipcode-binding protein. In Drosophila, Imp is part of a large, RNase-sensitive complex that is enriched in two polarized cell types, the developing oocyte and the neuron. Using time-lapse confocal microscopy, we establish that both dynein and kinesin contribute to the transport of GFP-Imp particles, and that regulation of transport in egg chambers appears to differ from that in neurons. In Drosophila, loss-of-function Imp mutations are zygotic lethal, and mutants die late as pharate adults. Imp has a function in Drosophila oogenesis that is not essential, as well as functions that are essential during embryogenesis and later development. Germline clones of Imp mutations do not block maternal mRNA localization or oocyte development, but overexpression of a specific Imp isoform disrupts dorsal/ventral polarity. We report here that loss-of-function Imp mutations, as well as Imp overexpression, can alter synaptic terminal growth. Our data show that Imp is transported to the neuromuscular junction, where it may modulate the translation of mRNA targets. In oocytes, where Imp function is not essential, we implicate a specific Imp domain in the establishment of dorsoventral polarity.
Figure 1. GFP-Imp Is Expressed in Ovaries and Neurons(A) In the ovary, GFP-Imp is detected within the germarium as early as stage 1 (arrow) and accumulates in the presumptive oocyte in early egg chambers. By stage 9–10, GFP-Imp is concentrated at the oocyte posterior pole (arrowheads). Bar, 25 μm.(B) GFP-Imp is enriched in the embryonic central nervous system (Bar, 25μm), and (C) in larval segmental axons and at the synapse (arrowhead). Bar, 5μm.(D) Schematic highlights the domain structure of Drosophila Imp compared to orthologs in human, chick, and Xenopus. RRM = RNA recognition motif. KH = hnRNP K homology domain.(E) The insertion of the PTT does not alter the level of Imp protein expression. Ovary extracts from wild type and GFP-Imp flies show similar levels of overall expression, as well as similar relative expression in supernatant (S) and pellet (P) fractions, as detected by an anti-Imp antibody.(F) GFP-Imp is present in a large, RNase-sensitive complex. GFP-Imp ovary extracts were fractionated over a 10%–40% sucrose gradient in the presence of RNase, or RNase inhibitors. Equal volumes of alternate fractions were analyzed by western blot using anti-GFP antibody. Lane L, sample of extract loaded on gradient. Fraction numbers are indicated above the lanes (bottom of gradient is fraction 1).
Figure 2. GFP-Imp Localization in the Oocyte Requires Microtubule Motor Function(A, A') GFP-Imp expression in wild type egg chambers is enriched in the oocyte and localizes to the posterior of the oocyte in stage 10.(B, B') Disruption of dynein using ΔGl overexpression (or the Dhc 6–6/6–12 transheterozygous mutant background; data not shown) produces an early aberrant accumulation of GFP-Imp to the oocyte anterior. However, the subsequent accumulation to the posterior pole is not affected.(C, C') In germline clones homozygous for the kinesin null mutation, khc27, GFP-Imp enrichment in the early oocyte is retained, but later accumulation at the posterior of the oocyte is lost. Bar (applies to all images), 10 μm.
Figure 3. Dynein and Kinesin Have Opposing Effects on GFP-Imp Motility in Nurse Cells(A) Three sequential images are separately pseudocolored red, green, or blue, and overlaid to highlight the frequency of GFP-Imp particle movements in the nurse cell cytoplasm in wild type and mutant backgrounds. Motility is easily discerned in wild type by the resolution of individual colors. After disruption of dynein (Dhc-) or dynactin (ΔGl), there is almost no movement; particles appear white, resulting from the superimposition of red, green, and blue. In contrast, disruption of kinesin I (Khc-) increases particle transport, as demonstrated by the increased visibility of individual colors. Bar, 10 μm. Genetic backgrounds shown include Dhc-, [Dhc6–6/Dhc6–12]; ΔGl, [UASp-ΔGl/+; nanos-GAL4/+]; and khc27, [khc27/ khc27 ] germline clones, as described in the text.(B) Motor mutations affect velocity as well as frequency of GFP-Imp transport events. Histogram illustrates the average velocities of GFP-Imp particles in nurse cells derived from wild type and mutant backgrounds. (1) wild type, (2) Dhc6–6/Dhc6–12, (3) UASp-ΔGl/+; nanos-GAL4, (4) khc27/ khc27germline clone, (5) wild type treated with colcemid, (6) wild type treated with cytochalasin D.
Figure 4. Imp Expression Affects Synaptic Terminal Size at the Neuromuscular Junction(A) Individual synaptic boutons are clearly labeled by the anti-CSP antibody. Wild type larval synapse is shown.(B) Bouton numbers are decreased in the Imp mutant combination, H44/H149.(C) Presynaptic overexpression of the UAS-RE transgene by elav-GAL4 (elav-GAL4/+; UAS-RE/+) significantly increases the number of boutons. Bar (applies to A–C), 50 μm.(D) Histogram compares relative synaptic terminal sizes of (1) wild type, (2) Imp H44/H149 mutant combination, and (3–5) overexpression of Imp transgenes, including (3) elav-GAL4/+; Imp-RE, (4) elav-GAL4/+; Imp-SD/+, and (5) elav-GAL4/+; Imp-KH/+. The synaptic terminal size was defined as the number of synaptic boutons normalized to the surface area of muscle 6/7, and the reference was set to 100 for the wild type control.(E) GFP-Imp is present at the synapse and accumulates along the membrane cortex of synaptic boutons (arrow). Bar, 5 μm.
Figure 5. Imp Mutations Do Not Disrupt the Nuclear Accumulation of pMad(A) In wild type, pMad is readily detected in the nuclei of motorneuron ganglia.(B) Nuclear uptake of pMad is retained in the Imp transheterozygous H44/H149 mutant background.(C) In contrast, pMad staining is abolished in the wit mutant. Bar, 10 μm.
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