Membrane Order Is a Key Regulator of Divalent Cation-Induced

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Membrane order is a key regulator of divalent cation-induced clustering of PI(3,5)P2 and PI(4,5)P2 Maria J. Sarmento, Ana Coutinho, Alexander A. Fedorov, Manuel Prieto, and Fabio Fernandes Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b00666 • Publication Date (Web): 29 Sep 2017 Downloaded from http://pubs.acs.org on October 2, 2017

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Membrane order is a key regulator of divalent cation-induced clustering of PI(3,5)P2 and PI(4,5)P2 Maria J. Sarmentoa,b, Ana Coutinhoa,c, Aleksander Fedorova, Manuel Prietoa, Fábio Fernandesa,d,* a.

Centro de Química-Física Molecular and Institute of Nanoscience and Nanotechnology, Instituto Superior Técnico, University of Lisbon, Lisbon, Portugal

b.

J. Heyrovský Inst. Physical Chemistry of the A.S.C.R. v.v.i., Prague, Czech Republic.

c.

Departamento de Química e Bioquímica, FCUL, University of Lisbon, Lisbon, Portugal

d.

UCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Campus da Caparica, Caparica, Portugal

*Corresponding author: E-mail address: [email protected]; Telephone number: +351 218 419 219; Fax number: +351 218 464 455


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ABSTRACT

Although the evidence for the presence of functionally important nanosized phosphorylated phosphoinositide (PIP)-rich domains within cellular membranes has accumulated, very limited information is available regarding the structural determinants for compartmentalization of these phospholipids. Here, we used a combination of fluorescence spectroscopy and microscopy techniques to characterize differences in divalent cation-induced clustering of PI(4,5)P2 and PI(3,5)P2. Through these methodologies we were able to detect differences in divalent cationinduced clustering efficiency and cluster size. Ca2+ induced PI(4,5)P2 clusters are shown to be significantly larger than the ones observed for PI(3,5)P2. Clustering of PI(4,5)P2 is also detected at physiological concentrations of Mg2+, suggesting that in cellular membranes, these molecules are constitutively driven to clustering by the high intracellular concentration of divalent cations. Importantly, it is shown that lipid membrane order is a key factor in the regulation of clustering for both PIP isoforms, with a major impact on cluster sizes. Clustered PI(4,5)P2 and PI(3,5)P2 are observed to present considerably higher affinity for more ordered lipid phases than the monomeric species or than PI(4)P, possibly reflecting a more general tendency of clustered lipids for insertion into ordered domains. These results support a model for the description of the lateral organization of PIPs in cellular membranes, where both divalent cation interaction and membrane order are key modulators defining the lateral organization of these lipids.


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INTRODUCTION

The most abundant phosphorylated phosphoinositide (PIP) in eukaryotic cells is phosphatidylinositol-4,5 bisphosphate (PI(4,5)P2). This phospholipid has been shown to control directly or indirectly a great number of cellular functions such as cell signaling1, protein2 and membrane trafficking3–5, actin cytoskeleton anchoring to the plasma membrane6, or cell adhesion7. Since several of these functions occur simultaneously and in many cases independently at the plasma membrane, they are thought to be maintained by the presence of functionally distinct pools of PI(4,5)P28,9. One of the mechanisms proposed to be responsible for the functional multiplicity of PI(4,5)P2 is the physical confinement of diffusion of this phospholipid within specialized regions of the plasma membrane8. In fact, despite its low and relatively constant concentration (~1 mol% of total membrane phospholipids in eukaryotic cells)10,11, PI(4,5)P2 is able to recruit specific proteins to particular sites of the plasma membrane at given times, thus controlling a variety of cell processes in time and space. The organization of PI(4,5)P2 in plasma membranes varies dramatically between cell types, with PI(4,5)P2 enriched heterogeneities observed at both the micron and the nanoscale 12–20. The mechanisms responsible for the complex membrane organization of PI(4,5)P2 are not entirely clear. While protein electrostatic interactions are very important21–24, the presence of membrane rafts or raft lipid components (cholesterol and sphingolipids) 17,18,25–27, have also been suggested to promote PI(4,5)P2 compartmentalization. Recently, another phosphorylated phosphoinositide, phosphatidylinositol-3,5 bisphosphate (PI(3,5)P2), was shown to segregate to ordered domains within yeast vacuole membranes, and to a distinct type of domains in endosomes/lysosomes of HeLa cells28.


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Additionally, the interaction of phosphatidylinositols with divalent cations29–35 has also been proposed to contribute to the lateral membrane organization of these phospholipids. Studies in model lipid membranes showed that the presence of divalent cations such as Ca2+ can induce formation of almost pure clusters of PI(4,5)P236.

While the lipid itself is homogeneously

distributed in liquid disordered membranes in the absence of divalent ions37, Ca2+ was shown to induce PI(4,5)P2 clustering in lipid monolayers32,36, large unilamellar vesicles (LUVs)38 and giant unilamellar vesicles (GUVs)33. Clustering is promoted through divalent cation-dependent bridging of two phospholipids via their phosphodiester groups39. Importantly, Ca2+ was recently shown to induce syntaxin-1 mesoscale clustering only in the presence of PI(4,5)P2

40

. This was interpreted to be the result of calcium establishing charge

bridges between Syntaxin-1/PI(4,5)P2 complexes and inducing Syntaxin-1 coalescence. This type of reorganization of membrane proteins due to specific Ca2+-PI(4,5)P2 interactions could be of major relevance in calcium signalling events. PIPs exhibit different relative Mg2+/Ca2+ affinities, with PI(4,5)P2 and PI(3,5)P2 exhibiting a preference for interaction with Ca2+ and Mg2+, respectively31,41 . These differences are likely a result of different charge densities in the headgroup of these lipids, with PI(3,5)P2’s negative potential spread over a larger area than PI(4,5)P2

41

. In fact, divalent cation-mediated attraction

of PIPs is dependent on lipid charge, phosphorylation positions and cation size

31,32,36

. A recent

study also showed that charge shielding of PI(4,5)P2 by different cations (including Mg2+) in membrane model systems and the plasma membrane, leads to considerable reduction in phospholipase C (PLC) activity, suggesting that divalent ions could regulate the amount of free PIP2 available for interaction with membrane proteins42. These results seem to indicate that differences in the phosphate position within the inositol ring (Figure 1) are enough to prompt


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different responses to the increase in the intracellular Ca2+ concentration after an external stimulus. Cells spend a significant amount of energy in order to maintain the intracellular calcium levels at a very low range (~100 nM), but concentrations in transient and localized calcium microdomains formed during certain cellular processes can be several orders of magnitude higher than these values (up to hundreds of µM in the vicinity of open calcium channels). On the other hand, Mg2+ cytosolic levels are in the low mM range, most of which (4-5 mM) is complexed, while a small fraction remains free (0.5–1mM)43. Since Mg2+ cytosolic concentration is several orders of magnitude higher than Ca2+, it is not clear if Ca2+ fluctuations can promote changes in the clustering of PIPs. The regulation of PI(4,5)P2 lateral organization by Ca2+ is of particular importance given the interplay of PI(4,5)P2, the PI(4,5)P2-derived second messengers diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3), and Ca2+ in diverse cell signalling processes 16,44–46.

Figure 1. Schematic representation of the inositol ring of PI(4)P, PI(3,4)P2 and PI(4,5)P2, focusing on the number and position of the phosphorylations.


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Previous work showed that Ca2+ favors partition of PI(4,5)P2 molecules towards cholesterolenriched domains, where clustering is more efficient30. Additionally, recent studies hinted that cholesterol could contribute to stabilize fluid PIP-enriched phases for several PIPs35,47. These results suggest that a concerted action of both divalent cations and membrane order in the regulation of PIP lateral organization is possible. In this work, we aim to compare the efficiency of divalent cation-induced clustering for different PIPs presenting distinct number and position of phosphorylations within the inositol ring – PI(4)P, PI(3,5)P2 and PI(4,5)P2. Importantly, we also focused on the role of membrane lipid composition in the clustering efficiencies of these phospholipids. Given the dynamic nature and nanoscale dimensions of Ca2+-induced PIP clustering, the detection and characterization of these clusters cannot be carried out through standard microscopy techniques. On the other hand, the application of spectroscopy methodologies using fluorescent analogues of PIPs were previously shown to be able to recover information on sizes and diffusion properties of these clusters, even at the very low physiological concentrations of both divalent cation and PIP30. Here, through the use of both fluorescent microscopy and spectroscopy tools, we were able to detect important differences in divalent cation-induced clustering efficiencies and cluster sizes for the different phosphoinositides, as well as significant differences in their partition towards ordered membrane phases in the presence of physiologically relevant calcium concentrations. This study reveals that the position of phosphorylations within the inositol ring of phosphoinositides, independently of protein interactions, is a key modulator of PIP lateral organization. These results support a new model for the description of PI(4,5)P2 lateral organization in cellular membranes, where apart from


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protein sequestration, both divalent cation interaction and membrane order actively contribute to its compartmentalization.

EXPERIMENTAL SECTION

Materials

1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero3-3-phospho-L-serine (POPS), N-palmitoyl-D-erythro-sphingosylphosphorylcholine (PSM), 1,2dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (DOPE-Cap-biotin), 1-oleoyl-2{6-[4-(dipyrrometheneboron

difluoride)butanoyl]

amino}

hexanoyl-sn-glycero-3-

phosphoinositol-4,5-bisphosphate (TF-PI(4,5)P2), 1,2-dioleoyl-sn-glycero-3-phospho-(1′-myoinositol-4′,5′-bisphosphate)

(PI(4,5)P2),

1-oleoyl-2-{6-[4-(dipyrrometheneboron

difluoride)butanoyl]amino}hexanoyl-sn-glycero-3-phosphoinositol-3,5-bisphosphate

(TF-

PI(3,5)P2), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol-3',5'-bisphosphate) (PI(3,5)P2), 1-oleoyl-2-{6-[4-(dipyrrometheneboron phosphoinositol-4-phosphate

difluoride)butanoyl]amino}

(TF-PI(4)P)

and

hexanoyl-sn-glycero-3-

1,2-dioleoyl-sn-glycero-3-phospho-(1'-myo-

inositol-4'-phosphate) (PI(4)P) were obtained from Avanti Polar Lipids (Alabaster, AL). Avidin from egg white and cholesterol (Chol) were from Sigma Chemical Co. (St. Louis,MO). Fluo-5N was obtained from Thermo Fisher Scientific (Waltham, MA, USA). All organic solvents were UVASOL grade from Merck Millipore (Darmstadt, Germany).


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Liposome preparation

Lipid stock solutions were prepared in chloroform, with the exception of all PIPs and TopFluor-PIPs (TF-PIPs) that were prepared in chloroform/methanol (2:1 v/v). The concentration of POPC, POPS, PSM, DOPE-Cap-biotin, PI(4,5)P2, PI(3,4)P2 and PI(4)P stock solutions were determined using an inorganic phosphate colorimetric quantification method48. Cholesterol concentration was determined by gravimetry (Mettler Toledo UMT2). Determination of fluorescent labeled PIP concentration was accomplished spectrophotometrically using ε(TF, 495 nm, methanol)= 80x103 M-1cm-1 49. For the preparation of multilamellar vesicles (MLVs), lipid mixtures composed of the adequate amount of lipids were prepared in chloroform to a final concentration of 0.25 mM. Total PIP amount (TF-PIP + unlabeled PIP) was always kept at 1 mol%. The solvent was slowly evaporated under a nitrogen flux and the resulting lipid films were left in vacuum overnight to ensure the complete removal of the organic solvent. Subsequently, the lipids were re-suspended in a 10 mM HEPES buffer (pH 7.4), containing 150 mM NaCl and the desired CaCl2 concentration. Freeze-thaw cycles (liquid nitrogen/water bath at 60 °C) were then performed to re-equilibrate and homogenize the samples. LUVs were prepared by extrusion of 0.5 mM MLVs. Extrusion was performed at 50 °C with an Avanti Mini-Extruder (Alabaster, AL) using 100 nm pore size polycarbonate membranes. As well as for MLVs, measurements were carried out at 0.25 mM total lipid concentration. For the formation of GUVs, all lipid mixtures were composed of POPC:PSM:Chol in different proportions and 0.2 mol% TF-PIP. Lipid solutions were prepared in chloroform (from lipid stock


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solutions) to a final lipid concentration of 1 mM. In order to immobilize GUVs, an avidin-coated surface was used for imaging purposes, and DOPE-Cap-biotin was included at a biotinylated lipid/total lipid ratio of 1/106

50

. GUVs were obtained by gel-assisted formation as previously

described51. Briefly, polyvinyl alcohol (PVA) was spread onto a microscope glass coverslip using a spin-coater (1200 rpm for 2 minutes) and was left drying for 15 minutes at 50 °C. The desired lipid mixture was then spread on the PVA surface. After evaporation of the solvent, a homemade chamber was assembled allowing gel-assisted GUV formation in 200 mM sucrose solution for 50-60 min at room temperature. Sucrose solutions were prepared with the desired CaCl2 concentration assuring the same calcium concentration in and out of the vesicles. After formation, GUVs were transferred to a µ-Slide from Ibidi (Munich, Germany) coated with avidin, and a 200 mM glucose solution was also added to the wells in order to increase GUV deposition and immobilization rate. All calcium concentrations were controlled using the calcium indicator Fluo-5N, following the manufacturer’s instructions. The buffer with no free calcium was prepared with 5 mM EDTA.

Steady-state fluorescence spectroscopy

Fluorescence measurements were carried out with a SLM-Aminco 8100 Series 2 spectrofluorimeter (Rochester, NY) with double excitation and emission monochromators (MC400), in right-angle geometry. The light source was a 450-W Xe arc lamp and the reference a Rhodamine B quantum counter solution. Quartz cuvettes (0.5×0.5 cm) from Hellma Analytics were used.


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Fluorescence intensities were corrected for inner filter effects as described elsewhere52. Steady-state fluorescence anisotropy, 〈〉 is defined as53:

〈〉 =

Eq. 1

where and are the steady-state vertical and horizontal components of fluorescence emission, respectively, with excitation accomplished with vertically polarized light. The components with horizontal excitation, and , are used to calculate the calibration factor ( = / ).

For the polarization of excitation and emission light, Glan-Thompson

polarizers were used. Subtraction of signal from blank samples was taken into account in all anisotropy components, as well as in the other fluorescence intensity measurements.

Time-resolved fluorescence spectroscopy

Fluorescence decay measurements were carried out via the Time-Correlated Single-Photon Couting (TCSPC) technique. Excitation was accomplished by a cavity-dumped dye laser (701-2, Coherent, delivering 5-6 ps pulses with ~40 nJ/pulse at 3.4 MHz) working with DCM (620-700 nm). The laser emission is doubled in a BBO crystal to deliver the pulses in 310-350 nm spectral range. TF-PIPs were excited at 335 nm, and fluorescence emission was acquired at 505 nm. The emission wavelength was selected by a Jobin Yvon HR320 monochromator (Horiba Jobin Ivon Inc.). 0.5x0.5 cm quartz cuvettes from Hellma Analytics were used. Decays from blank samples were also acquired and photon counts were found to be negligible. The fluorescence intensity decays were described by a sum of exponentials:


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) = ∑ −/ )

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Eq. 2

where is the normalized amplitude and is the component lifetime. For the TF probe, usually 3 exponentials are required for the proper fit of Eq. 2 to the data. The amplitudeweighted average lifetime is defined by:

̅ = ∑

Eq. 3

Data analysis was performed with the TRFA software (Scientific Software Technologies Center, Minsk, Belarus) based on the Levenberg-Marquardt algorithm. The goodness of the fit was judged from the experimental ! , weighted residuals and autocorrelation plot. In every analysis, ! was less than 1.3 and both residuals and autocorrelation were randomly distributed around zero.

Confocal laser scanning microscopy

Fluorescence imaging was performed on a Leica TCS SP5 (Leica Microsystems CMS GmbH, Mannheim, Germany) inverted confocal microscope (DMI6000). A 63x amplification apochromatic water immersion objective with NA=1.2 (Zeiss, Jena, Germany) was used for all measurements. An Argon laser was used for excitation. After GUV immobilization, images were acquired at 100 Hz, exciting the TF fluorophore at 488 nm and collecting emission between 500 and 550 nm. GUVs exhibiting phase coexistence were measured at the equatorial plane for


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determination of partition coefficients (KP) of labeled phospholipids according to the following equation54:

')(

#$% = 〈 &) ( 〉 〈 *

)*+ ,+ -̅( 〉 + ,( -̅+ & ')

Eq. 4

where . ) is the fluorescence intensity value measured in a position of a equatorial slice of a confocal image of a GUV within phase i. /' is a factor quantifying the extent of photoselection observed in position and for phase i. 0 is the brightness of the fluorophore in the corresponding phase and was determined from fluorescence spectroscopy measurements in LUVs with compositions corresponding to each of the pure lipids phases, according to a published phase diagram55. 1̅ is the average lipid molecular area characteristic of each lipid phase. The average area per lipid of POPC and Chol were considered to be 62 and 26 Å2, respectively56,57. The average area per PSM molecule was estimated to be 43 Å2 on the basis of literature results for the pure lipid58 and for the impact of cholesterol on the condensation of similar sphingomyelin lipids59. For the POPC:PSM:Chol compositions on the extremes of the tie-line chosen in this study (71.6:23.3:5 and 25.4:34.8:39.8 mol/mol/mol), the ratio of average area per lipid in each phase (1̅23 ⁄1̅24 ) was estimated to be 0.74. Data analysis was carried out using homemade software developed in a Matlab environment (Mathworks, Natick, MA).

RESULTS AND DISCUSSION Methodological approach


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We have previously shown that the sizes of PI(4,5)P2 clusters at physiological concentrations of both PI(4,5)P2 and Ca2+ are well below the resolution of even super-resolution fluorescence imaging techniques30. Since such small and dynamic nanoscale structures can only be efficiently characterized through spectroscopic tools, fluorescent analogues of PIPs, labeled with a TopFluor moiety at the sn-2 acyl chain (to prevent any modifications of the inositol ring) and with an oleoyl chain at sn-1 (TF-PIPs) were used here to monitor PIP lateral organization (Figure 2).

Figure 2. Structure of the different TopFluor-labelled phosphatidylinositols.

In this work, we monitored both fluorescence self-quenching (for detection of PIP cluster formation) and homoFRET (for evaluation of differences in cluster size) between TF-PIP analogues to have independent and complementary information on the PIP clustering process. TopFluor is a hydrophobic derivative of BODIPY, and in fact, BODIPY-labeled lipids are known to exhibit self-quenching due to the formation of non-fluorescent species when two


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analogues are in very close proximity60. Such dimers can originate from ground state interaction (static quenching) or from statistical collisions (dynamic quenching)61,62. When clustered, BODIPY-labeled lipids exhibit considerably more efficient self-quenching due to the increased average proximity between fluorophores and this can be monitored through measurements of fluorescence intensity or lifetime. This method has been successfully used to monitor proteininduced clustering of PI(4,5)P222,63. Additionally, due to its small Stokes shift, TF and BODIPY fluorophores are capable of undergoing energy migration (Energy Homotransfer) to other TF-labeled molecules present at a close range (Förster radius R0 = 57 Å64). When the local density of TF-labeled analogues increases (as upon enrichment in a lipid cluster or domain), FRET becomes more efficient, leading to fluorescence depolarization (decrease of fluorescence anisotropy). Although monitoring the self-quenching of TF-PIP or BODIPY-PIP is an efficient tool for detection of PIP clustering, it cannot provide information on the dimensions of such structures, since self-quenching requires molecular contact to occur and the quantum yield of each PIP analogue will only be dependent on the concentration of other analogues in its immediate environment. On the other hand, given the large R0 of TF-PIP, homoFRET can occur between analogues at distances over 100 Å, and the efficiency of this process becomes critically dependent on cluster size (and the resulting changes in the number of acceptor molecules within the cluster)

65

. In this way, quantification of homoFRET through the measurement of

fluorescence anisotropies can be used to monitor changes in cluster size. It should be stressed that according to the Perrin equation

66

, and as discussed in detail

elsewhere46, dynamic self-quenching of TF-PIP analogues would, in the absence of FRET, lead to an increase in anisotropy values (as the fluorophore has less time for rotation during the


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excited sate). In this way, observation of a concomitant depolarization of TF-PIP fluorescence upon clustering is reflective of high homoFRET efficiency within clusters 30.

The mono-phosphorylated PI(4)P does not exhibit clustering in the presence of Ca2+ or highly ordered membranes

The number of phosphate groups at the inositol ring of PIPs determines the net charge of their head groups, and can therefore modulate their interaction with divalent cations such as Ca2+. Different PIPs are thus expected to present different susceptibilities for divalent cation-induced clustering. In order to evaluate if nanoscale calcium-induced clustering (as previously observed for PI(4,5)P2 at physiological concentrations30) can also occur for mono-phosphorylated PIPs, fluorescence anisotropy measurements were carried out for TF-PI(4)P within POPC unilamellar lipid vesicles. For comparison, experiments with TF-PI(4,5)P2 were also performed in parallel. The total PIP concentration was kept at 1 mol% in all samples through the inclusion of unlabelled PIP. In the absence of Ca2+, both TF-PI(4)P and TF-PI(4,5)P2 display very similar fluorescence depolarization profiles, although the fluorescence anisotropy is marginally higher for TFPI(4,5)P2 (Figure 3). In addition, the fluorescence anisotropy of both TF-PI(4)P2 and TFPI(4,5)P2 varies with the concentration of TF-PIP as predicted by theoretical models for FRET between non-interacting and homogeneously distributed molecules 67.


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Figure 3. Ca2+-induced PIP clustering is not observed for the mono-phosphorylated PI(4)P in liquid disordered membranes. Steady-state fluorescence anisotropy measurements were performed using LUVs of POPC containing different molar fractions of A) TF-PI(4)P and B) TF-PI(4,5)P2. The respective unlabeled PIP was added to achieve 1 mol% of total PIP in each sample. Vesicles were prepared in the presence (~100 µM Ca2+, orange) and absence (5 mM EDTA, blue) of free Ca2+. The decrease in fluorescence anisotropy for higher concentrations of PI(4)P fluorescent analogues is independent of Ca2+ and is a result of FRET between noninteracting and homogeneously distributed TF-PI(4)P. On the other hand, PI(4,5)P2 clustering in the presence of Ca2+ is easily identified by the decrease in fluorescence anisotropy (increase in FRET) relative to the results obtained in the absence of divalent ions. Statistical significance is clear and was confirmed by a two-way ANOVA analysis (p

) values were measured in

GUVs composed of POPC:PSM:Chol (45.1:29.9:25) to achieve 58 % lo phase, and containing 0.2 mol% of (A) TF-PI(4,5)P2, (B) TF-PI(3,5)P2 and (C) TF-PI(4)P. Imaging was accomplished by exciting the samples at 488 nm and collecting emission at 500-550 nm. (A) examples of confocal images obtained in the presence and absence of Ca2+. Scale bars represent 5 µm. Lipid phases are discriminated on the basis of photoselection differences, as this effect is more prominent in lo phases. ld phases corresponded always to the brighter lipid phase due to extensive


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2 /2?

PIP2 self-quenching in lo phases in the presence of divalent cations (Figure 5 and 6). (B) #=>

values were determined from TF fluorescence intensity in each phase using Eq.4, in the presence of 100 µM Ca2+ (orange), 5 mM Mg2+ (blue), and in the absence of divalent cations (grey). Error bars represent the standard error of the mean (*p

values, evidencing a high preference for the more disordered phase (Figure 12B). In the

case of PI(4)P, where no clustering occurs either in the presence or absence of Ca2+ or Mg2+, the addition of the divalent cations did not result in significant changes in its partition coefficient. These results also confirm that the number of phosphorylations in the inositol ring has no impact on the membrane partition properties of PIPs in the absence of divalent ions. However, for both PIP2 isomers, divalent cation-induced clustering led to a dramatic increase in the affinity for the lo phase. In the presence of 100 µM Ca2+, both TF-PI(4,5)P2 and TF-PI(3,5)P2 still present in 2 /2?

average a moderate preference for ld domains (#=>

2 /2?

(#=>

close to unity and TF-PI(4,5)P2 showing moderate preference for the lo phase

>1). From these results, it seems likely that on cellular membranes, clustered PI(4,5)P2

will be directed to more ordered domains, and that this could be a general principle driving PI(4,5)P2 lateral organization.


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In this work, only PIPs with single unsaturated fatty acids were used, while the majority of PI(4,5)P2 in mammalians contains a polyunsaturated (C20:4) fatty acid chain. For this reason, it is likely that within the plasma membrane, the affinity of clustered polyunsaturated PI(4,5)P2 for ordered domains is to some extent lower than the values reported here.

CONCLUSIONS

Here, we confirm that while both PI(3,5)P2 and PI(4,5)P2 form very high density clusters in model

membranes

in

the

presence

of

physiological

concentrations

of

Ca2+

and

phosphatidylinositol, the dimensions of PI(4,5)P2 clusters are found to be significantly larger than the ones obtained for its isomer. It is possible that this difference reflects the higher density of charge in PI(4,5)P241, contributing for the formation of stable PI(4,5)P2 clusters in the plasma membrane, where the concentration of this phospholipid has already been determined to be 1-2 orders of magnitude higher than the average plasma membrane concentration in certain cell types13,16,71. Similar PI(4,5)P2 clustering is observed in the presence of Mg2+, although higher concentrations of the divalent cation are required. On the other hand, in POPC membranes, PI(3,5)P2 clustering with Mg2+ appears to be negligible or generating only small aggregates. PI(4,5)P2 clustering was evaluated in both MLVs or LUVs, and no differences in PI(4,5)P2 clustering were observed between these two model membrane systems with different membrane curvatures, suggesting that membrane curvature is not a critical factor regulating PIP2 clustering. Membrane order is shown here to heavily influence the properties of divalent cation-dependent PI(4,5)P2 clusters. In fact, the dimensions of these clusters are shown to be highly dependent on


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both membrane composition and temperature. Importantly, large PI(4,5)P2 clusters are obtained even at low sphingomyelin levels. As seen here, the miscibility of monomeric PI(4,5)P2 species in ordered membranes is considerably lower than in more disordered lipid phases, due to the presence of an unsaturated acyl-chain and large headgroup. This is likely to contribute to stabilize larger clusters of these lipids in ordered membranes. Ca2+-induced PI(3,5)P2 cluster sizes are not significantly dependent on membrane order. On the other hand, in the presence of Mg2+, PI(3,5)P2 cluster sizes are significantly larger for more ordered membranes. For PI(4,5)P2, the presence of both Ca2+ and Mg2+ at high concentrations, had no significant cumulative effect on PIP2 clustering over the one observed with each of the divalent cations separately. These results suggest that PI(4,5)P2 in particular is likely to be constitutively driven to clustering while not complexed with membrane protein partners. In combination with sequestering by membrane proteins and the presence of physical barriers for diffusion, this clustering is likely to contribute to the stabilization of PI(4,5)P2 enriched patches at particular sites of the plasma membrane, since diffusion coefficients of PI(4,5)P2 decrease when the lipid forms these aggregates46. Also, it is shown that PIP2 molecules display significantly less affinity for disordered domains after divalent cation-induced clustering. The role of multivalent complexation of other plasma membrane functional lipids in stabilizing their enrichment in ordered domains has already been demonstrated72. It is likely that insertion of poly-phosphorylated PIPs in more ordered cellular membrane domains is similarly stabilized by complexation through divalent cations. These results also suggest that within the plasma membrane, the distribution of free polyphosphorylated PIPs, namely PI(4,5)P2 given its relative abundance, is constantly distinct of that of its precursor PI(4)P, even though they present equivalent relative concentrations and only


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differ by one phosphorylation at the inositol ring. Such differentiation could help direct PI(4,5)P2 to areas of the plasma membrane where it is required to regulate specific effector proteins, and where the concentration of PI(4,5)P2 is increased several times relative to other less specific polyanionic lipids such as PI(4)P. In this case, clustered PI(4,5)P2 would present dramatically different affinity for incorporation in membrane domains with increased ordering, unlike the monomeric PI(4)P. The results shown here strongly support the idea that the properties and lateral organization of PIP2 in cellular membranes are not mirrored by PIP2 in model membranes in the absence of divalent cations. In particular, the potential of PIP2 clusters (with Ca2+ or Mg2+) to shield polyphosphorylated PIPs from interactions with membrane proteins is not entirely clear and requires further investigation. Regarding this issue, recent results suggest that charge shielding of PI(4,5)P2 by divalent cations plays an important regulatory role for several PI(4,5)P2 dependent functions

42,73–75

. The considerable differences in organization of polyphosphoinositides in the

presence of physiologically relevant concentrations of divalent cations are likely to have an important impact in complex membrane processes dependent on PIPs such as endocytosis or phagocytosis, where different PIPs exhibit dramatically different lateral segregation profiles in the plasma membrane. During these processes, the action of kinases and phosphatases will not only modify protein-lipid interactions, but will also shift the distribution of free PIPs from monomeric to clustered or vice versa.

Supporting Information. Comparison of Ca2+-induced PI(4,5)P2 clustering in MLVs and LUVs (Figure S2). Dependence of TF-PI(3,5)P2 and TF-PI(4,5)P2 fluorescence lifetime with cholesterol concentration for different SM content (Figure S2). Dependence of

TF-PI(3,5)P2 and TF-


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PI(4,5)P2 ∆r values with cholesterol concentration for different SM content (Figure S3). Description of models for homoFRET within homogeneous lipid membranes and within lipid clusters. This material is available free of charge via the Internet at http://pubs.acs.org.

Corresponding Author *E-mail: [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENTS FF acknowledges the Portuguese National Funding Agency FCT (Fundação para a Ciência e a Tecnologia) through Researcher Contract No. IF/00386/2015. MJS and AF were recipient of fellowships

from

FCT

(SFRH/BD/80575/2011,

SFRH/BPD/111301/2015).

Authors

acknowledge funding by FCT project reference FAPESP/20107/2014. This project has received funding from European Structural & Investment Funds through the COMPETE Programme and from National Funds through FCT under the Programme grant SAICTPAC/0019/2015.


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Figure 1 254x190mm (300 x 300 DPI)


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Figure 2 254x190mm (300 x 300 DPI)


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Figure 3 310x118mm (150 x 150 DPI)


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Figure 4 113x100mm (220 x 220 DPI)


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Figure 5 338x190mm (96 x 96 DPI)


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Figure 6 196x179mm (150 x 150 DPI)


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Figure 7 187x190mm (150 x 150 DPI)


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Figure 8 130x108mm (150 x 150 DPI)


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Figure 9 170x78mm (150 x 150 DPI)


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Figure 10 201x146mm (150 x 150 DPI)


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Figure 11 118x130mm (150 x 150 DPI)


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Figure 12 360x170mm (300 x 300 DPI)


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