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First published online 11 March 2008
doi: 10.1242/jcs.024927

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Depletion of plasma membrane PtdIns(4,5)P₂ reveals essential roles for phosphoinositides in flagellar biogenesis

Ho-Chun Wei¹, Janet Rollins^2,*, Lacramioara Fabian¹, Madeline Hayes³, Gordon Polevoy¹, Christopher Bazinet² and Julie A. Brill^1,3,

¹ Program in Developmental and Stem Cell Biology, The Hospital for Sick Children, Toronto, Ontario M5G 1L7, Canada
² Department of Biological Sciences, St John's University, Queens, NY 11439, USA
³ Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada

View larger version (114K): [in this window] [in a new window]   Fig. 1. SigD expression depletes plasma membrane PtdIns(4,5)P2. (A) Simplified PtdIns pathway. PtdIns (PI) can be phosphorylated by PtdIns 4-kinases (PI4K), such as Drosophila Fwd, to yield PtdIns(4)P [PI4P]. PtdIns(4)P is phosphorylated by PtdIns(4)P 5-kinases (PIP5K), such as Drosophila Sktl, to produce PtdIns(4,5)P2 (PIP2). PtdIns(4,5)P2 is hydrolyzed by phospholipase C (PLC), generating the second messengers inositol trisphosphate (InsP3, IP3) and diacylglycerol (DAG). InsP3 can lead to release of Ca2+ from intracellular stores (via the InsP3 receptor) and to synthesis of other inositol polyphosphates, such as inositol hexakisphosphate (InsP6, IP6). Alternatively, PtdIns(4,5)P2 can be sequentially dephosphorylated by 5-phosphatases (5-P'ase), including Salmonella SigD, to regenerate PtdIns(4)P, which in turn can be dephosphorylated by 4-phosphatases (4-P'ase), such as Drosophila Sac1, to produce PtdIns. (B-E') Phase-contrast (B-E) and corresponding fluorescence (B'-E') images of male germ cells expressing PLC-PH-GFP (B,C,E) or PLC-PH-RFP (D), which bind PtdIns(4,5)P2. (B') PtdIns(4,5)P2 is found in the plasma membrane (arrow) of wild-type male germ cells. (C') SigD expression diminishes PtdIns(4,5)P2 at the plasma membrane and causes accumulation of PLC-PH-GFP puncta in the cytoplasm (arrow). (D') Co-expression of Sktl partially restores plasma membrane PtdIns(4,5)P2 (arrow), although some cells exhibit non-specific PLC-PH-RFP localization in the cytoplasm. (E') Expression of SigDdead has no effect on PtdIns(4,5)P2 accumulation. (F-G') Phase-contrast (F,G) and corresponding fluorescence (F',G') images of male germ cells expressing RFP-PH-FAPP to detect PtdIns(4)P. (F') PtdIns(4)P localizes to the Golgi in spermatocytes (arrowhead) and to the acroblast in spermatids (arrow). (G') SigD expression increases PtdIns(4)P levels in both spermatocytes (arrowhead) and spermatids (arrow). Note that, although the exposure times for F' and G' were identical, the contrast in F' was increased by 50% to make the PtdIns(4)P localization more obvious. Scale bars: 20 µm.

View larger version (120K): [in this window] [in a new window]   Fig. 2. PtdIns(4,5)P2 depletion inhibits sperm tail formation and causes defects in microtubule organization. (A-D) Phase-contrast micrographs of whole Drosophila testes. (A) A wild-type testis has many rope-like cysts of elongating spermatids (arrow). (B) Expression of SigD causes the accumulation of ovoid spermatid cysts (arrow). (C) Co-expression of Sktl with SigD restores spermatid elongation (arrow). (D) Expression of SigDdead has no effect on sperm tail formation (arrow). (E-I) Confocal fluorescence micrographs of spermatids expressing β-tubulin-GFP (β-tub-GFP, green) and stained for DNA (blue). (E,H) Wild-type spermatids. (E) β-tub-GFP is incorporated into elongating axonemes and perinuclear forked microtubule (MT) arrays (arrow). (H) After individualization, β-tub-GFP is found in prominent perinuclear puncta (arrows) and axonemes (inset, magnified cyst). (F,G,I) SigD-expressing spermatids. (F) Early elongating cyst showing β-tub-GFP in axonemes (arrowhead) and large diffuse perinuclear MT arrays (arrow). (G) Cysts with tangled tails of β-tub-GFP (arrowhead). (I) Later-stage cysts show poorly aligned MTs (arrowhead) and puncta dissociated from nuclei (arrows) (inset, magnified cyst). Scale bars: 50 µm (A-D), 20 µm (E-I).

View larger version (78K): [in this window] [in a new window]   Fig. 3. PtdIns(4,5)P2 depletion affects peripheral basal body proteins. (A-C) Analysis of the core basal body protein GFP-PACT. (A,B) Fluorescence micrographs of (A) wild-type and (B) SigD-expressing spermatids, showing the morphology of GFP-PACT-containing basal bodies (arrows). Note that some SigD-expressing cells are multinucleate and contain four basal bodies (B, arrow). (C) Analysis of the length of the GFP-PACT signal in wild-type (wt) versus PtdIns(4,5)P2-depleted (SigD) spermatids indicates no significant difference in the length of GFP-PACT-containing basal bodies (P=0.0957). (D-F) Fluorescence micrographs of spermatids stained for the basal-body-associated protein Centrosomin (Cnn, green, arrows) and dynein heavy chain (DHC, red, arrowheads). (D) In wild-type spermatids, Cnn localizes to puncta that are tightly associated with DHC in the nuclear envelope (arrow and arrowhead). (E) In SigD-expressing spermatids, Cnn localizes to diffuse comet-shaped structures that are not always tightly associated with the nuclear envelope (arrow). DHC localization appears normal (arrowhead). (F) Spermatids co-expressing SigD and Sktl exhibit normal DHC and Cnn localization. (G-I) Spermatids stained for -tubulin (-tub, red) and DNA (blue). (G) Early and late (inset) wild-type spermatids. Note that early spermatids have barely discernible -tub foci (arrow), whereas late spermatids exhibit prominent -tub puncta that closely associate with nuclei (one per nucleus). (H) Early and late (inset) SigD-expressing spermatids show aberrant -tub accumulation (arrow) and loose association of -tub and nuclei (inset). (I) Early and late (inset) spermatids expressing SigD and Sktl show restored -tub localization (arrow). Scale bars, 10 µm (A,B,D-F), 20 µm (G-I).

View larger version (96K): [in this window] [in a new window]   Fig. 4. PtdIns(4,5)P2 depletion affects axoneme outgrowth. (A) Diagrams showing Unc-GFP (green) localization in developing spermatids. (i) Unc-GFP is found at the basal body in early round spermatids (arrow). Nucleus (white circle) and mitochondrial derivatives (black circle), which are wrapped together at this stage, are shown. (ii-iv) Elongating spermatids. (ii) Protein body stage. (iii) Later stage of elongation. (iv) Late stage of elongation showing nuclear shaping. During elongation, Unc-GFP is found at the basal body (iv, arrow) and at the elongating end (iv, arrowhead). Not to scale. (B-H') Phase-contrast (B-H) and corresponding fluorescence (B'-H') micrographs of spermatids expressing Unc-GFP (green) and stained for DNA (blue). (B,D,F) Wild-type spermatids. (B') Unc-GFP (arrow) is present at the basal body of early round spermatids. (D') As spermatids elongate, Unc-GFP signals split into two parts, a cylindrical structure at the basal body (arrows) and a prominent dot associated with the growing end (arrowheads). In addition, a weak Unc-GFP signal can be seen along the entire length of the elongating sperm tail (inset, between arrow and arrowhead). (F') In later stages, Unc-GFP signals are clearly observed at opposite ends of the elongating cyst [arrow, nuclei (mostly on a different focal plane); arrowhead, growing end]. Note that the contrast of this image was increased by 30% to make the localization of Unc-GFP more obvious. (C,E,G,H) Spermatids expressing SigD. (C') Unc-GFP localization appears comet-shaped (arrow) in SigD-expressing spermatids. Association of Unc-GFP with nuclear DNA is also disrupted. (E') In a rare elongated SigD-expressing cyst, Unc-GFP splits into two parts (arrow, arrowhead), and a weak signal along the sperm tail can be seen. (G') SigD cyst at the protein body stage. Note the failure of Unc-GFP to split into two signals. (H') Later-stage cyst with Unc-GFP signals scattered in the cytoplasm. Scale bars: 10 µm (B,C,G), 20 µm (D,E), 50 µm (F,H). Inset in D is approximately the same scale as G.

View larger version (190K): [in this window] [in a new window]   Fig. 5. PtdIns(4,5)P2 depletion affects axoneme architecture. (A-C) Transmission electron micrographs (TEMs) showing the distribution of axonemes (arrowheads) in spermatid cysts. (A) Wild-type cysts have many axonemes with a regular 9 + 2 arrangement (inset, magnified axoneme). (B) Spermatid cysts expressing SigD have few axonemes, and these axonemes have aberrant MT organization (inset). (C) Co-expression of Sktl with SigD restores axonemes of normal architecture (inset). (D-I) TEMs showing axonemal architecture and axial membranes. Major (m1) and minor (m2) mitochondrial derivatives are indicated. (D,G) Wild-type axonemes. (D) Wild-type spermatids have doublet MTs (arrow) surrounded by axial membranes (triple arrows) adjacent to the two mitochondrial derivatives. (G) Axonemes from later-stage spermatids develop accessory MTs that are centripetally displaced from the outer doublet MTs (inset, arrow). (E,H) Axonemes from SigD-expressing spermatids. (E) Note the presence of triplet MTs (arrow). (H) Axial membranes (triple arrows) fail to ensheath axonemes that are falling apart (arrowhead). No SigD axonemes with accessory MTs were observed. (F,I) Co-expression of Sktl with SigD restored axonemal doublets and accessory MTs (arrow, I); however, occasional defects in doublet orientation were observed (arrow, F). Scale bars: 500 nm (A-C), 100 nm (D-I).

View larger version (149K): [in this window] [in a new window]   Fig. 6. PtdIns(4,5)P2 depletion interferes with basal body docking to the nuclear envelope. (A-K) TEMs showing developing basal bodies from spermatids at different stages of development. Nuclei (n) and basal bodies (arrows) are indicated. (A-D) Wild-type spermatids. (A) Longitudinal section through an early-stage spermatid showing attachment of the basal body to the nuclear envelope. (B) Longitudinal section through the nucleus, basal body and centriolar adjunct of an intermediate-stage spermatid. (C) Cross-section through an intermediate-stage spermatid showing the position of the centriole and centriolar adjunct with respect to the two mitochondrial derivatives (m1, m2). (D) Longitudinal section through the nucleus and basal body region of an advanced spermatid. (E-H) Spermatids from testes expressing SigD. (E,F) Longitudinal sections of early-stage SigD-expressing spermatids showing defects in orientation and attachment of the basal body and the centriolar adjunct to the nuclear envelope. (G) Cross-section through an intermediate-stage SigD-expressing spermatid showing dissociation of the basal body and centriolar adjunct from the nuclear envelope. (H) Basal body, enlarged centriolar adjunct and axoneme from a later-stage SigD-expressing spermatid. (I-K) Axonemes from testes co-expressing SigD and Sktl. (I) Longitudinal section through the nucleus and the centriolar adjunct of an intermediate-stage spermatid showing proper orientation of the basal body with respect to the nuclear envelope. (J) Longitudinal section through the nucleus and basal body region of an advanced spermatid showing the basal body embedded in the nuclear envelope. (K) Longitudinal section showing a basal body and corresponding axoneme not embedded in the nuclear membrane. Scale bars: 500 nm.