PI4P-containing organelles localize to the midzone during cytokinesis
The requirement for Fwd in spermatocyte cytokinesis suggested that PI4P and secretory trafficking likely play critical roles in this process. To determine if PI4P associates with secretory cargo at the midzone, we examined dividing cells coexpressing an RFP fusion to the PI4P marker PH-FAPP (RFP-PH-FAPP; Dowler et al., 2000
; Wei et al., 2008
) and a secreted GFP (sGFP; Pfeiffer et al., 2000
) in real-time imaging experiments (Wong et al., 2005
) (). RFP-PH-FAPP and sGFP were concentrated at the poles of the cell in early telophase and were also associated with parafusorial membranes that extend along the length of the meiotic spindle (; t = 00:00, arrowheads). In later stages of telophase, sGFP and RFP-PH-FAPP continued to localize to the poles of the cell, but also colocalized in a small number of puncta at the midzone (; arrows, t = 05:00). The association of these markers at the midzone appeared to increase as cleavage progressed (; t = 11:00–18:00). Colocalization of PI4P and secretory cargo at the midzone suggested that Fwd catalytic activity is required for cytokinesis.
Figure 1. PI4P-containing organelles localize at the midzone during cytokinesis. Phase–contrast (phase) and corresponding fluorescence time-lapse images of a dividing spermatocyte expressing RFP-PH-FAPP as a marker for PI4P and sGFP as a secretory marker. (more ...)
PI 4-kinase activity is required to restore fertility to fwd mutant flies
To determine if catalytic activity of Fwd, that is, the conversion of PI to PI4P, is crucial for cytokinesis and male fertility, we generated transgenic flies expressing GFP fused to either wild-type Fwd (GFP-Fwd) or a kinase-dead (KD) version of the protein (GFP-FwdKD
). The latter carries an amino acid substitution (D1191A) analogous to a mutation (D656A) that abolishes catalytic function of the human and bovine enzymes in vitro (Godi et al., 1999
; see Fig. S1
). Examination of live squashed preparations of wild-type early round spermatids revealed a 1:1 correspondence of haploid nuclei to mitochondrial derivatives of similar size (; Fuller, 1993
). In contrast, fwd
mutant testes exhibited multinucleate spermatids containing either two or four haploid nuclei accompanied by an enlarged mitochondrial derivative (, arrowheads), indicating a failure of meiotic cytokinesis (; Brill et al., 2000
). Importantly, GFP-Fwd completely rescued the cytokinesis defects and infertility of fwd
null males (; and not depicted). Surprisingly, expression of GFP-FwdKD
partially rescued the cytokinesis defect of fwd
mutant males, causing an increase in the frequency of normal haploid spermatids ().
Figure 2. Drosophila Fwd and mammalian PI4Kβ rescue the cytokinesis defect of fwd mutant males. (A) Phase–contrast micrographs of early round haploid spermatids from wild-type, fwd mutant (fwd/Df), and fwd mutant flies expressing GFP-Fwd or GFP-Fwd (more ...)
To determine if mammalian PI4Kβ can substitute for Fwd, we generated flies expressing either bovine or hemagglutinin (HA)-tagged human PI4Kβ (bPI4K or hPI4K). Remarkably, the mammalian enzymes fully rescued the cytokinesis defects and infertility of fwd
mutant males (), suggesting that the role of Fwd has been evolutionarily conserved. In parallel, we also expressed a KD version of hPI4K (Godi et al., 1999
). As with GFP-FwdKD
, expression of hPI4KKD
partially rescued the fwd
cytokinesis defect. Both the fly and human KD constructs decreased the probability of cytokinesis failure by roughly 40% (), indicating that PI4Kβ may have noncatalytic as well as catalytic functions in cytokinesis. However, because neither FwdKD
rescued the infertility of fwd
mutant males, the enzyme's catalytic activity and hence, PI4P, is required for male germ cell development.
Fwd is required for Golgi accumulation of PI4P
To characterize the subcellular distribution of PI4P, we examined spermatocytes coexpressing RFP-PH-FAPP with Fws-GFP, a known Golgi marker (Farkas et al., 2003
). In wild type, 82% of RFP-PH-FAPP puncta (n
= 361) colocalized with Fws-GFP (), indicating that PI4P is found on Golgi membranes. Interestingly, the RFP-PH-FAPP signal was often both overlapping and adjacent to Fws-GFP, indicating that Fws and PI4P may be concentrated in distinct regions of the Golgi.
Figure 3. PI4P and Fwd colocalize on Golgi membranes. (A and B) Phase–contrast (phase) and corresponding fluorescence micrographs of live squashed wild-type or fwd mutant (fwd/Df) spermatocytes expressing RFP-PH-FAPP (PI4P) with Fws-GFP (A) or with GFP-Fwd (more ...)
To test whether Fwd was required for Golgi morphology or for PI4P accumulation on Golgi membranes, we examined Fws-GFP, Lva (Sisson et al., 2000
), and RFP-PH-FAPP. The distribution of Fws-GFP () and Lva (not depicted) appeared unaltered in fwd
mutant spermatocytes, suggesting Golgi morphology was normal. However, the distribution of RFP-PH-FAPP was severely affected by loss of fwd
. RFP-PH-FAPP appeared cytoplasmic and diffuse, although occasional bright puncta colocalized with Fws-GFP (, arrows). Note that we do not expect RFP-PH-FAPP to become completely diffuse because PH-FAPP binds Golgi-localized Arf1 (Lemmon, 2008
) and may also bind PI4P synthesized by other PI4Ks. Thus, Fwd is required for accumulation of normal levels of PI4P on Golgi membranes.
To examine localization of Fwd, we compared the distribution of GFP-Fwd with that of PI4P. In wild-type (not depicted) and fwd mutant spermatocytes, GFP-Fwd colocalized with RFP-PH-FAPP on Golgi membranes (, arrows); 93% of GFP-Fwd puncta colocalized with RFP-PH-FAPP (n = 212) and 86% of RFP-PH-FAPP puncta colocalized with GFP-Fwd (n = 228). GFP-Fwd also colocalized with RFP-PH-FAPP at the poles of dividing cells in telophase but, unlike PI4P (), appeared to be absent from the midzone in squashed preparations (, arrowheads). Indeed, time-lapse imaging of dividing wild-type spermatocytes expressing GFP-Fwd confirmed this result (). Thus, Fwd colocalizes with its lipid product on Golgi membranes but not at the midzone during cytokinesis.
To determine if Fwd catalytic activity influences the distribution of Fwd or PI4P, we examined colocalization of GFP-FwdKD and RFP-PH-FAPP in wild-type versus fwd mutant cells (). In wild type, GFP-FwdKD localization was indistinguishable from that of GFP-Fwd, and RFP-PH-FAPP appeared unaffected, indicating that GFP-FwdKD does not interfere with PI4P biosynthesis (not depicted). Localization of GFP-FwdKD appeared normal in fwd mutant spermatocytes. However, the intensity of RFP-PH-FAPP-containing puncta appeared reduced (, arrows), similar to that observed in fwd mutant spermatocytes lacking FwdKD (). Because GFP-Fwd and GFP-FwdKD were expressed at similar levels (not depicted), Fwd kinase activity promotes normal accumulation of PI4P.
Fwd is required for colocalization of PI4P and sGFP at the midzone
Based on the phenotype of fwd mutant spermatocytes, we predicted that Fwd is required for midzone localization of PI4P. To test this, we examined live squashed preparations of dividing spermatocytes for localization of RFP-PH-FAPP and sGFP (). A majority of wild-type cells showed RFP-PH-FAPP and sGFP at the midzone in late telophase (43/45 cells). In contrast, few telophase fwd spermatocytes showed midzone RFP-PH-FAPP or sGFP and, when present, their localization appeared diffuse (5/19 cells) (, arrows). In addition, the numbers of RFP-PH-FAPP and sGFP-positive puncta were substantially reduced in fwd mutant cells. This was particularly obvious in highly squashed cells in which the plasma membrane was no longer associated with the midzone (, arrows). In wild type, RFP-PH-FAPP and sGFP precisely colocalized in multiple puncta in the cleavage plane, as well as in individual puncta in other regions of the cytoplasm. In fwd, the few sGFP-containing puncta at the midzone failed to colocalize with RFP-PH-FAPP and sGFP rarely colocalized with RFP-PH-FAPP in other regions of the cell. Thus, Fwd is required for formation and localization of PI4P-containing secretory organelles during late stages of cytokinesis.
Figure 4. fwd is required for midzone accumulation of PI4P and sGFP. (A and B) Phase–contrast (phase) and corresponding fluorescence micrographs of live preparations of dividing wild-type and fwd mutant (fwd/Df) spermatocytes coexpressing sGFP and RFP-PH-FAPP (more ...)
The failure of PI4P and sGFP to localize properly in dividing fwd
mutant cells suggested a potential defect in distribution of Golgi membranes during cytokinesis. To test this, we examined localization of the Golgi proteins in dividing cells. Fws-GFP and Lva localized to the poles in both wild-type and fwd
mutant spermatocytes (). However, in fwd
mutant cells, Fws-GFP appeared less punctate and more diffuse, and Lva-containing puncta appeared smaller. In addition, as previously reported (Giansanti et al., 2006
), Lva was also found in the midzone of fwd
mutant cells (, arrows). Thus, proper Golgi organization during cytokinesis appears to require Fwd.
Fwd binds and colocalizes with Rab11
partially rescued the fwd
mutant phenotype, we hypothesized that Fwd might directly bind other proteins as part of its function in cytokinesis. To identify Fwd binding partners, we performed yeast two-hybrid tests on Drosophila
homologues of proteins previously reported to bind yeast or mammalian PI4Kβ. As bait, we used full-length and truncated versions of Fwd. As prey, we tested Frq (the homologue of S. cerevisiae
Frq1p), Mlc-c (myosin essential light chain, the homologue of Schizosaccharomyces pombe
Cdc4p), and Rab11, together with several related proteins. For the Frq experiments, we also used as bait a stretch of amino acids from yeast Pik1 that binds mammalian Ncs-1 (Strahl et al., 2003
). Frq and the related protein Nca strongly bound this portion of Pik1, but not full-length or truncated versions of Fwd (unpublished data). Fwd also did not interact with Mlc-c or the related myosin regulatory light chain Sqh (unpublished data). In contrast, Rab11 showed a strong two-hybrid interaction with Fwd (). The binding was specific to Rab11, as Rab5 and Rab7 failed to interact in the same assay.
Figure 5. Fwd binds and colocalizes with the recycling endosome regulator Rab11. (A) Yeast two-hybrid assays. (Left) Patches of yeast cells cotransformed with Fwd or FwdKD bait plasmids together with one of several prey plasmids (vector alone, Rab5, Rab7, Rab11, (more ...)
To determine if the interaction between Fwd and Rab11 depended on either the catalytic activity of Fwd or the GTP-binding state of Rab11, we tested mutated versions of Fwd and Rab11. Both Fwd and FwdKD interacted with wild-type Rab11, Rab11Q70L (GTP-bound), and Rab11S25N (GDP-bound). Wild-type Fwd exhibited stronger binding interactions with Rab11 than did FwdKD (), but their interactions showed similar trends: both Fwd and FwdKD appeared to bind slightly better to Rab11Q70L than to Rab11S25N, and the weakest binding in each case was with wild-type Rab11. Importantly, the lack of strong preferential binding of Fwd to activated (GTP-bound) Rab11 suggested that Fwd, rather than being a Rab11 effector, might regulate Rab11 in vivo.
To verify the binding of Fwd to Rab11, we performed coIP experiments on fly proteins expressed in mammalian tissue culture cells (). Flag-tagged Rab11 and HA-tagged Fwd or FwdKD were expressed in COS-7 cells, alone or in combination. IPs with anti-Flag antibody yielded HA-Fwd or HA-FwdKD only in the presence of Flag-Rab11. Reciprocally, IPs with anti-HA antibody yielded Flag-Rab11 only in the presence of HA-Fwd or HA-FwdKD. Thus, Drosophila Fwd, like mammalian PI4Kβ, binds the recycling endosome regulator Rab11.
The binding of Fwd to Rab11 suggested the two proteins might colocalize in vivo. To test this, we examined developing male germ cells expressing CFP and YFP fusions to Fwd (CFP-Fwd) and Rab11 (YFP-Rab11). Rab11 and Fwd colocalized at the Golgi in spermatocytes (, arrows) and at the poles of dividing cells (not depicted). In spermatocytes, 91.9% of CFP-Fwd puncta (n = 280) colocalized with YFP-Rab11 and 82.3% of YFP-Rab11 puncta (n = 318) colocalized with CFP-Fwd. In dividing cells, Rab11 also localized to structures where Fwd was not evident, including parafusorial membranes and puncta at the midzone (see next section). Thus, Fwd localizes to a subset of Rab11-positive structures before and during meiotic cytokinesis.
Rab11 colocalizes with PI4P and its localization depends on Fwd
Because the distribution of Rab11 appeared similar to that of PI4P, we examined flies coexpressing GFP-Rab11 with RFP-PH-FAPP. In spermatocytes, GFP-Rab11 tightly colocalized with RFP-PH-FAPP on Golgi membranes (, top panels, arrows). 98.2% of GFP-Rab11 puncta (n = 171) colocalized with RFP-PH-FAPP and 77.4% of RFP-PH-FAPP–positive puncta (n = 217) were positive for GFP-Rab11. Strikingly, GFP-Rab11 and RFP-PH-FAPP also colocalized to parafusorial membranes and at the midzone of dividing cells (, bottom panels, arrows). Thus, Rab11 is found primarily on PI4P-containing organelles, including those found in the midzone during cytokinesis.
Figure 6. Rab11 localizes to PI4P-containing organelles and its localization during cytokinesis requires fwd. (A and B) Phase–contrast (phase) and corresponding fluorescence micrographs of live squashed spermatocytes expressing GFP-Rab11 and RFP-PH-FAPP. (more ...)
To test whether Fwd regulates Rab11 localization, we examined GFP-Rab11 in wild-type versus fwd mutant flies. In fwd mutant spermatocytes, GFP-Rab11 localized to puncta resembling those found in wild-type cells (compare , top panels, arrows), and showed partial colocalization with residual RFP-PH-FAPP. However, in dividing spermatocytes and elongating spermatids, the distribution of GFP-Rab11 was clearly aberrant (compare , bottom panels, arrows). In contrast to wild type, GFP-Rab11 appeared completely delocalized during cytokinesis in fwd mutant cells. Moreover, at elongating stages, GFP-Rab11 concentrated in unusual linear elements along the elongating spermatid tails rather than the uniform distribution seen in wild type (, arrowheads). Thus, Fwd is required for proper localization of Rab11 during and after cytokinesis.
To further characterize the effect of Fwd on Rab11 activity during cytokinesis, we examined telophase cells from wild-type and fwd mutant flies for localization of Rab11 and its effector Nuf using specific antibodies (). In wild type (n = 85), Rab11 (62.3%) and Nuf (94.1%) localized in puncta at the midzone (, top panels, arrows). However, in fwd mutant cells, localization of Rab11 and Nuf was more variable (, middle panels, arrows). Occasionally, Rab11 (17.7%) and Nuf (19.1%) were concentrated at the midzone (n = 68 cells). However, in the majority of cells, Rab11 was undetectable (82.3%) and Nuf was either undetectable (76.5% cells) or localized to only a portion of the midzone (4.4%; not depicted). Thus, Fwd is required for localization of Rab11 and its effector Nuf in dividing cells.
Figure 7. Rab11 acts downstream of fwd to promote completion of cytokinesis. (A) Phase–contrast (phase) and fluorescence images of dividing spermatocytes stained for Rab11 (green), Nuf (red), and DNA (blue). Arrows indicate the midzone. Colocalization of (more ...)
Rab11 acts downstream of Fwd during cytokinesis
If Rab11 acts downstream of Fwd, overexpression of activated Rab11 should suppress the cytokinesis defect caused by mutations in fwd. Indeed, overexpression of Rab11Q70L, but not wild-type Rab11 or dominant-negative Rab11 (Rab11S25N), partially suppressed the fwd cytokinesis defect (; Rab11S25N, not depicted). Moreover, overexpression of activated Rab11 (Rab11Q70L) restored Nuf localization to the midzone in 82.6% of dividing cells (n = 23) (, bottom panels, arrows). Thus, activated Rab11 can partially compensate for loss of fwd.