Phosphatidylinositol 4,5-Bisphosphate Influences PIN Polarization by Controlling Clathrin-Mediated Membrane Trafficking in Arabidopsis

The functions of the minor phospholipid phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P₂] during vegetative plant growth remain obscure. Here, we targeted two related phosphatidylinositol 4-phosphate 5-kinases (PI4P 5-kinases) PIP5K1 and PIP5K2, which are expressed ubiquitously in Arabidopsis thaliana. A pip5k1 pip5k2 double mutant with reduced PtdIns(4,5)P₂ levels showed dwarf stature and phenotypes suggesting defects in auxin distribution. The roots of the pip5k1 pip5k2 double mutant had normal auxin levels but reduced auxin transport and altered distribution. Fluorescence-tagged auxin efflux carriers PIN-FORMED (PIN1)–green fluorescent protein (GFP) and PIN2-GFP displayed abnormal, partially apolar distribution. Furthermore, fewer brefeldin A–induced endosomal bodies decorated by PIN1-GFP or PIN2-GFP formed in pip5k1 pip5k2 mutants. Inducible overexpressor lines for PIP5K1 or PIP5K2 also exhibited phenotypes indicating misregulation of auxin-dependent processes, and immunolocalization showed reduced membrane association of PIN1 and PIN2. PIN cycling and polarization require clathrin-mediated endocytosis and labeled clathrin light chain also displayed altered localization patterns in the pip5k1 pip5k2 double mutant, consistent with a role for PtdIns(4,5)P₂ in the regulation of clathrin-mediated endocytosis. Further biochemical tests on subcellular fractions enriched for clathrin-coated vesicles (CCVs) indicated that pip5k1 and pip5k2 mutants have reduced CCV-associated PI4P 5-kinase activity. Together, the data indicate an important role for PtdIns(4,5)P₂ in the control of clathrin dynamics and in auxin distribution in Arabidopsis.     

Phosphatidylinositol-4,5-bisphosphate influences Nt-Rac5-mediated cell expansion in pollen tubes of Nicotiana tabacum

The regulation of pollen tube growth by the phospholipid phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2) ) is not well understood. The Arabidopsis genome encodes two type A phosphatidylinositol-4-phosphate (PI4P) 5-kinases, PIP5K10 and PIP5K11, which are exclusively expressed in pollen and produce PtdIns(4,5)P(2) in vitro. Fluorescence-tagged PIP5K10 and PIP5K11 localized to lateral subapical plasma membrane microdomains in tobacco pollen tubes in a pattern closely resembling the distribution of PtdIns(4,5)P(2,) with the exception of notably weaker association at the extreme apex. Overexpression of PIP5K10 or PIP5K11 in tobacco pollen tubes resulted in severe tip swelling and altered actin fine structure similar to that reported for overexpression of tobacco Nt-Rac5, a monomeric GTPase known to regulate the actin cytoskeleton. Increased sensitivity of Arabidopsis pip5k10 pip5k11 double mutant pollen tubes to Latrunculin B (LatB) further supports a role for type A PI4P 5-kinases in controlling the actin cytoskeleton. Despite the disruption of both its type A PI4P 5-kinases, the pip5k10 pip5k11 double mutant was fertile, indicating that one of the remaining type B PI4P 5-kinase isoforms might be functionally redundant with PIP5K10 and PIP5K11. Antagonistic effects of PIP5K11 and the Nt-Rac5-specific guanine nucleotide dissociation inhibitor, Nt-RhoGDI2, on tip swelling observed in coexpression-titration experiments indicate a link between PtdIns(4,5)P(2) and Rac-signaling in pollen tubes. The data suggest that type A PI4P 5-kinases influence the actin cytoskeleton in pollen tubes in part by counteracting Nt-RhoGDI2, possibly contributing to the control of the pool of plasma membrane-associated Nt-Rac5.     

Arabidopsis phosphatidylinositol 4-phosphate 5-kinase genes PIP5K7, PIP5K8, and PIP5K9 are redundantly involved in root growth adaptation to osmotic stress

Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) produces phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P₂), a signaling phospholipid critical for various cellular processes in eukaryotes. The Arabidopsis thaliana genome encodes 11 PIP5K genes. Of these, three type B PIP5K genes, PIP5K7, PIP5K8, and PIP5K9, constitute a subgroup highly conserved in land plants, suggesting that they retain a critical function shared by land plants. In this study, we comprehensively investigated the biological functions of the PIP5K7–9 subgroup genes. Reporter gene analyses revealed their preferential expression in meristematic and vascular tissues. Their YFP-fusion proteins localized primarily to the plasma membrane in root meristem epidermal cells. We selected a mutant line that was considered to be null for each gene. Under normal growth conditions, neither single mutants nor multiple mutants of any combination exhibited noticeable phenotypic changes. However, stress conditions with mannitol or NaCl suppressed main root growth and reduced proximal root meristem size to a greater extent in the pip5k7pip5k8pip5k9 triple mutant than in the wild type. In root meristem epidermal cells of the triple mutant, where plasma membrane localization of the PtdIns(4,5)P₂ marker P24Y is impaired to a large extent, brefeldin A body formation is retarded compared with the wild type under hyperosmotic stress. These results indicate that PIP5K7, PIP5K8, and PIP5K9 are not required under normal growth conditions, but are redundantly involved in root growth adaptation to hyperosmotic conditions, possibly through the PtdIns(4,5)P₂ function promoting plasma membrane recycling in root meristem cells.     

Phosphatidylinositol 4,5-bisphosphate is important for stomatal opening

Previously, we demonstrated that a protein that binds phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] inhibits both light-induced stomatal opening and ABA-induced stomatal closing. The latter effect is due to a reduction in free PtdIns(4,5)P(2), decreasing production of inositol 1,4,5-trisphosphate and phosphatidic acid by phospholipases C and D. However, it is less clear how PtdIns(4,5)P(2) modulates stomatal opening. We found that in response to white light irradiation, the PtdIns(4,5)P(2)-binding domain GFP:PLCdelta1PH translocated from the cytosol into the plasma membrane. This suggests that the level of PtdIns(4,5)P(2) increases at the plasma membrane upon illumination. Exogenously administered PtdIns(4,5)P(2) substituted for light stimuli, inducing stomatal opening and swelling of guard cell protoplasts. To identify PtdIns(4,5)P(2) targets we performed patch-clamp experiments, and found that anion channel activity was inhibited by PtdIns(4,5)P(2). Genetic analyses using an Arabidopsis PIP5K4 mutant further supported the role of PtdIns(4,5)P(2) in stomatal opening. The reduced stomatal opening movements exhibited by a mutant of Arabidopsis PIP5K4 (At3g56960) was countered by exogenous application of PtdIns(4,5)P(2). The phenotype of reduced stomatal opening in the pip5k4 mutant was recovered in lines complemented with the full-length PIP5K4. Together, these data suggest that PIP5K4 produces PtdIns(4,5)P(2) in irradiated guard cells, inhibiting anion channels to allow full stomatal opening.     

Complex regulation of Arabidopsis AGR1/PIN2-mediated root gravitropic response and basipetal auxin transport by cantharidin-sensitive protein phosphatases

Polar auxin transport, mediated by two distinct plasma membrane-localized auxin influx and efflux carrier proteins/complexes, plays an important role in many plant growth and developmental processes including tropic responses to gravity and light, development of lateral roots and patterning in embryogenesis. We have previously shown that the Arabidopsis AGRAVITROPIC 1/PIN2 gene encodes an auxin efflux component regulating root gravitropism and basipetal auxin transport. However, the regulatory mechanism underlying the function of AGR1/PIN2 is largely unknown. Recently, protein phosphorylation and dephosphorylation mediated by protein kinases and phosphatases, respectively, have been implicated in regulating polar auxin transport and root gravitropism. Here, we examined the effects of chemical inhibitors of protein phosphatases on root gravitropism and basipetal auxin transport, as well as the expression pattern of AGR1/PIN2 gene and the localization of AGR1/PIN2 protein. We also examined the effects of inhibitors of vesicle trafficking and protein kinases. Our data suggest that protein phosphatases, sensitive to cantharidin and okadaic acid, are likely involved in regulating AGR1/PIN2-mediated root basipetal auxin transport and gravitropism, as well as auxin response in the root central elongation zone (CEZ). BFA-sensitive vesicle trafficking may be required for the cycling of AGR1/PIN2 between plasma membrane and the BFA compartment, but not for the AGR1/PIN2-mediated root basipetal auxin transport and auxin response in CEZ cells.     

The Arabidopsis Phosphatidylinositol Phosphate 5-Kinase PIP5K3 is a key regulator of root hair tip growth

Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] functions as a site-specific signal on membranes to promote cytoskeletal reorganization and membrane trafficking. Localization of PtdIns(4,5)P2 to apices of growing root hairs and pollen tubes suggests that it plays an important role in tip growth. However, its regulation and mode of action remain unclear. We found that Arabidopsis thaliana PIP5K3 (for Phosphatidylinositol Phosphate 5-Kinase 3) encodes a phosphatidylinositol 4-phosphate 5-kinase, a key enzyme producing PtdIns(4,5)P2, that is preferentially expressed in growing root hairs. T-DNA insertion mutations that substantially reduced the expression of PIP5K3 caused significantly shorter root hairs than in the wild type. By contrast, overexpression caused longer root hairs and multiple protruding sites on a single trichoblast. A yellow fluorescent protein (YFP) fusion of PIP5K3, driven by the PIP5K3 promoter, complemented the short-root-hair phenotype. PIP5K3-YFP localized to the plasma membrane and cytoplasmic space of elongating root hair apices, to growing root hair bulges, and, notably, to sites about to form root hair bulges. The signal was greatest in rapidly growing root hairs and quickly disappeared when elongation ceased. These results provide evidence that PIP5K3 is involved in localizing PtdIns(4,5)P2 to the elongating root hair apex and is a key regulator of the machinery that initiates and promotes root hair tip growth.     

Type B Phosphatidylinositol-4-Phosphate 5-Kinases Mediate Arabidopsis and Nicotiana tabacum Pollen Tube Growth by Regulating Apical Pectin Secretion

Phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P₂] occurs in the apical plasma membrane of growing pollen tubes. Because enzymes responsible for PtdIns(4,5)P₂ production at that location are uncharacterized, functions of PtdIns(4,5)P₂ in pollen tube tip growth are unresolved. Two candidate genes encoding pollen-expressed Arabidopsis thaliana phosphatidylinositol-4-phosphate 5-kinases (PI4P 5-kinases) of Arabidopsis subfamily B were identified (PIP5K4 and PIP5K5), and their recombinant proteins were characterized as being PI4P 5-kinases. Pollen of T-DNA insertion lines deficient in both PIP5K4 and PIP5K5 exhibited reduced pollen germination and defects in pollen tube elongation. Fluorescence-tagged PIP5K4 and PIP5K5 localized to an apical plasma membrane microdomain in Arabidopsis and tobacco (Nicotiana tabacum) pollen tubes, and overexpression of either PIP5K4 or PIP5K5 triggered multiple tip branching events. Further studies using the tobacco system revealed that overexpression caused massive apical pectin deposition accompanied by plasma membrane invaginations. By contrast, callose deposition and cytoskeletal structures were unaltered in the overexpressors. Morphological effects depended on PtdIns(4,5)P₂ production, as an inactive enzyme variant did not produce any effects. The data indicate that excessive PtdIns(4,5)P₂ production by type B PI4P 5-kinases disturbs the balance of membrane trafficking and apical pectin deposition. Polar tip growth of pollen tubes may thus be modulated by PtdIns(4,5)P₂ via regulatory effects on membrane trafficking and/or apical pectin deposition.     

Cooperation of phosphoinositides and BAR domain proteins in endosomal tubulation

Phosphorylated derivatives of phosphatidylinositol (PtdIns) regulate many intracellular events, including vesicular trafficking and actin remodeling, by recruiting proteins to their sites of function. PtdIns(4,5)-bisphosphate [PI(4,5)P2] and related phosphoinositides are mainly synthesized by type I PtdIns-4-phosphate 5-kinases (PIP5Ks). We found that PIP5K induces endosomal tubules in COS-7 cells. ADP-ribosylation factor (ARF) 6 has been shown to act upstream of PIP5K and regulate endocytic transport and tubulation. ARF GAP with coiled-coil, ankyrin repeat, and pleckstrin homology domains 1 (ACAP1) has guanosine triphosphatase-activating protein (GAP) activity for ARF6. While there were few tubules induced by the expression of ACAP1 alone, numerous endosomal tubules were induced by coexpression of PIP5K and ACAP1. ACAP1 has a pleckstrin homology (PH) domain known to bind phosphoinositide and a Bin/amphiphysin/Rvs (BAR) domain that has been reported to detect membrane curvature. Truncated and point mutations in the ACAP1 BAR and PH domains revealed that both BAR and PH domains are required for tubulation. These results suggest that two ARF6 downstream molecules, PIP5K and ACAP1, function together in endosomal tubulation and that phosphoinositide levels may regulate endosomal dynamics.     

BYPASS1-LIKE regulates lateral root initiation via exocytic vesicular trafficking-mediated PIN recycling in Arabidopsis

Auxin and auxin-mediated signaling pathways are known to regulate lateral root development. Although exocytic vesicle trafficking plays an important role in recycling the PIN-FORMED (PIN) auxin efflux carriers and in polar auxin transport during lateral root formation, the mechanistic details of these processes are not well understood. Here, we demonstrate that BYPASS1-LIKE (B1L) regulates lateral root initiation via exocytic vesicular trafficking-mediated PIN recycling in Arabidopsis thaliana. b1l mutants contained significantly more lateral roots than the wild type, primarily due to increased lateral root primordium initiation. Furthermore, the auxin signal was stronger in stage I lateral root primordia of b1l than in those of the wild type. Treatment with exogenous auxin and an auxin transport inhibitor indicated that the lateral root phenotype of b1l could be attributed to higher auxin levels and that B1L regulates auxin efflux. Indeed, compared to the wild type, C-terminally green fluorescent protein-tagged PIN1 and PIN3 accumulated at higher levels in b1l lateral root primordia. B1L interacted with the exocyst, and b1l showed defective PIN exocytosis. These observations indicate that B1L interacts with the exocyst to regulate PIN-mediated polar auxin transport and lateral root initiation in Arabidopsis.     

Arabidopsis PLDzeta2 regulates vesicle trafficking and is required for auxin response

Phospholipase D (PLD) and its product, phosphatidic acid (PA), play key roles in cellular processes, including stress and hormonal responses, vesicle trafficking, and cytoskeletal rearrangements. We isolated and functionally characterized Arabidopsis thaliana PLDzeta2, which is expressed in various tissues and enhanced by auxin. A PLDzeta2-defective mutant, pldzeta2, and transgenic plants deficient in PLDzeta2 were less sensitive to auxin, had reduced root gravitropism, and suppressed auxin-dependent hypocotyl elongation at 29 degrees C, whereas transgenic seedlings overexpressing PLDzeta2 showed opposite phenotypes, suggesting that PLDzeta2 positively mediates auxin responses. Studies on the expression of auxin-responsive genes and observation of the beta-glucuronidase (GUS) expression in crosses between pldzeta2 and lines containing DR5-GUS indicated that PLDzeta2, or PA, stimulated auxin responses. Observations of the membrane-selective dye FM4-64 showed suppressed vesicle trafficking under PLDzeta2 deficiency or by treatment with 1-butanol, a PLD-specific inhibitor. By contrast, vesicle trafficking was enhanced by PA or PLDzeta2 overexpression. Analyses of crosses between pldzeta2 and lines containing PIN-FORMED2 (PIN2)-enhanced green fluorescent protein showed that PLDzeta2 deficiency had no effect on the localization of PIN2 but blocked the inhibition of brefeldin A on PIN2 cycling. These results suggest that PLDzeta2 and PA are required for the normal cycling of PIN2-containing vesicles as well as for function in auxin transport and distribution, and hence auxin responses.     

Impacts of iron on phosphate starvation-induced root hair growth in Arabidopsis

In Arabidopsis, phosphate starvation (-Pi)-induced responses of primary root and lateral root growth are documented to be correlated with ambient iron (Fe) status. However, whether and how Fe participates in -Pi-induced root hair growth (RHG) remains unclear. Here, responses of RHG to different Fe concentrations under Pi sufficiency/deficiency were verified. Generally, distinct dosage effects of Fe on RHG appeared at both Pi levels, due to the generation of reactive oxygen species. Following analyses using auxin mutants and the phr1 mutant revealed that auxin and the central regulator PHR1 are required for Fe-triggered RHG under −Pi. A further proteomic study indicated that processes of vesicle trafficking and auxin synthesis and transport were affected by Fe under −Pi, which were subsequently validated by using a vesicle trafficking inhibitor, brefeldin A, and an auxin reporter, R2D2. Moreover, vesicle trafficking-mediated recycling of PIN2, an auxin efflux transporter, was notably affected by Fe under -Pi. Correspondingly, root hairs of pin2 mutant displayed attenuated responses to Fe under -Pi. Together, we propose that Fe affects auxin signalling probably by modulating vesicle trafficking, chiefly the PIN2 recycling, which might work jointly with PHR1 on modulating -Pi-induced RHG.     

Arabidopsis PCaP2 modulates the phosphatidylinositol 4,5‐bisphosphate signal on the plasma membrane and attenuates root hair elongation

Phosphatidylinositol 4,5‐bisphosphate [PtdIns(4,5)P₂] serves as a subcellular signal on the plasma membrane, mediating various cell‐polarized phenomena including polar cell growth. Here, we investigated the involvement of Arabidopsis thaliana PCaP2, a plant‐unique plasma membrane protein with phosphoinositide‐binding activity, in PtdIns(4,5)P₂ signaling for root hair tip growth. The long‐root‐hair phenotype of the pcap2 knockdown mutant was found to stem from its higher average root hair elongation rate compared with the wild type and to counteract the low average rate caused by a defect in the PtdIns(4,5)P₂‐producing enzyme gene PIP5K3. On the plasma membrane of elongating root hairs, the PCaP2 promoter‐driven PCaP2–green fluorescent protein (GFP), which complemented the pcap2 mutant phenotype, overlapped with the PtdIns(4,5)P₂ marker 2xCHERRY‐2xPH^PLC in the subapical region, but not at the apex, suggesting that PCaP2 attenuates root hair elongation via PtdIns(4,5)P₂ signaling on the subapical plasma membrane. Consistent with this, a GFP fusion with the PCaP2 phosphoinositide‐binding domain PCaP2^N23, root hair‐specific overexpression of which caused a low average root hair elongation rate, localized more intense to the subapical plasma membrane than to the apical plasma membrane similar to PCaP2–GFP. Inducibly overexpressed PCaP2–GFP, but not its derivative lacking the PCaP2^N23 domain, replaced 2xCHERRY‐2xPH^PLC on the plasma membrane in root meristematic epidermal cells, and suppressed FM4‐64 internalization in elongating root hairs. Moreover, inducibly overexpressed PCaP2 arrested an endocytic process of PIN2–GFP recycling. Based on these results, we conclude that PCaP2 functions as a negative modulator of PtdIns(4,5)P₂ signaling on the subapical plasma membrane probably through competitive binding to PtdIns(4,5)P₂ and attenuates root hair elongation.     

Regulation of phosphatidylinositol 4,5-bisphosphate levels and its roles in cytoskeletal re-organization and malignant transformation.

It is well known that phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) plays important roles not only as a precursor lipid for generating second messengers but also as a regulator of cytoskeletal re-organization. The last step of PtdIns(4,5)P2 synthesis is catalyzed by PtdIns monophosphate(PIP) kinase. So far, three type I PIP kinases(alpha, beta, and gamma), which phosphorylate PtdIns(4) to PtdIns(4,5)P2, and three type II PIP kinases(alpha, beta, gamma), which phosphorylate PtdIns(5)P to PtdIns(4,5)P2 have been found. On the other hand, several inositolpolyphosphate 5-phosphatases which convert PtdIns(4,5)P2 to PtdIns(4) are known. Among them, synaptojanin, which associates with tyrosine kinase receptors through an adaptor protein, Ash/Grb2, in response to growth factors, is capable of hydrolyzing PtdIns(4,5)P2 bound to actin regulatory proteins, resulting in actin filament re-organization downstream of tyrosine kinases.     

Plasma membrane nano-organization specifies phosphoinositide effects on Rho-GTPases and actin dynamics in tobacco pollen tubes

Pollen tube growth requires coordination of cytoskeletal dynamics and apical secretion. The regulatory phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) is enriched in the subapical plasma membrane of pollen tubes of Arabidopsis thaliana and tobacco (Nicotiana tabacum) and can influence both actin dynamics and secretion. How alternative PtdIns(4,5)P₂ effects are specified is unclear. In tobacco pollen tubes, spinning disc microscopy (SD) reveals dual distribution of a fluorescent PtdIns(4,5)P₂-reporter in dynamic plasma membrane nanodomains vs. apparent diffuse membrane labeling, consistent with spatially distinct coexisting pools of PtdIns(4,5)P₂. Several PI4P 5-kinases (PIP5Ks) can generate PtdIns(4,5)P₂ in pollen tubes. Despite localizing to one membrane region, the PIP5Ks AtPIP5K2-EYFP and NtPIP5K6-EYFP display distinctive overexpression effects on cell morphologies, respectively related to altered actin dynamics or membrane trafficking. When analyzed by SD, AtPIP5K2-EYFP associated with nanodomains, whereas NtPIP5K6-EYFP localized diffusely. Chimeric AtPIP5K2-EYFP and NtPIP5K6-EYFP variants with reciprocally swapped membrane-associating domains evoked reciprocally shifted effects on cell morphology upon overexpression. Overall, active PI4P 5-kinase variants stabilized actin when targeted to nanodomains, suggesting a role of nanodomain-associated PtdIns(4,5)P₂ in actin regulation. This notion is further supported by interaction and proximity of nanodomain-associated AtPIP5K2 with the Rho-GTPase NtRac5, and by its functional interplay with elements of Rho of plants signaling. Plasma membrane nano-organization may thus aid the specification of PtdIns(4,5)P₂ functions to coordinate cytoskeletal dynamics and secretion.

The apical plasma membrane of pollen tubes contains nanodomains where the regulatory phospholipid PtdIns(4,5)P₂ exerts a stabilizing effect on the actin cytoskeleton.     

Overexpression of murine phosphatidylinositol 4-phosphate 5-kinase type Ibeta disrupts a phosphatidylinositol 4,5 bisphosphate regulated endosomal pathway

The type I phosphatidylinositol 4-phosphate 5-kinases (PI4P5K) phosphorylate phosphatidylinositol 4-phosphate [PI(4)P] to produce phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. PI(4,5)P2 has been implicated in signal transduction, receptor mediated endocytosis, vesicle trafficking, cytoskeletal structure, and membrane ruffling. However, the specific type I enzymes associated with the production of PI(4,5)P2 for the specific cellular processes have not been rigorously defined. Murine PI4P5K type Ibeta (mPIP5K-Ibeta) was implicated in receptor mediated endocytosis through the isolation of a truncated and inactive form of the enzyme that blocked the ligand-dependent downregulation of the colony-stimulating factor-1 receptor. The present study shows that enforced expression of mPIP5K-Ibeta in 293T cells resulted in the accumulation of large vesicles that were linked to an endosomal pathway. Similar results were obtained after the expression of the PI(4,5)P2-binding pleckstrin homology (PH) domain of phospholipase-Cdelta (PLC-delta). Analysis of the conserved domains of mPIP5K-Ibeta led to the identification of dimerization domains in the N- and C-terminal regions. Enforced expression of the individual dimerization domains interfered with the proper subcellular localization of mPIP5K-Ibeta and the PLC-delta-PH domain and blocked the accumulation of the endocytic vesicles induced by these proteins. In addition to regulating early steps in endocytosis, these results suggest that mPIP5K-Ibeta acts through PI(4,5)P2 to regulate endosomal trafficking and/or fusion.     

PtdIns(4,5)P2 functions at the cleavage furrow during cytokinesis

Phosphoinositides play important roles in regulating the cytoskeleton and vesicle trafficking, potentially important processes at the cleavage furrow. However, it remains unclear which, if any, of the phosphoinositides play a role during cytokinesis. A systematic analysis to determine if any of the phosphoinositides might be present or of functional importance at the cleavage furrow has not been published. Several studies hint at a possible role for one or more phosphoinositides at the cleavage furrow. The best of these are genetic data identifying mutations in phosphoinositide-modifying enzymes (a PtdIns(4)P-5-kinase in S. pombe and a PI-4-kinase in D. melanogaster) that interfere with cytokinesis. The genetic nature of these experiments leaves questions as to how direct may be their contribution to cytokinesis. Here we show that a single phosphoinositide, PtdIns(4,5)P2, specifically accumulates at the furrow. Interference with PtdIns(4,5)P2 interferes with adhesion of the plasma membrane to the contractile ring at the furrow. Finally, four distinct interventions to specifically interfere with PtdIns(4,5)P2 each impair cytokinesis. We conclude that PtdIns(4,5)P2 is present at the cleavage furrow and is required for normal cytokinesis at least in part because of a role in adhesion between the contractile ring and the plasma membrane.