Stomatal guard cells are unique as a plant cell model and, because of the depth of knowledge now to hand on ion transport and its regulation, serve as an excellent model for the analysis of stimulus-response coupling in higher plants. Parallel controls – mediated by Ca2+, H+protein kinases and
phosphatases — regulate the gating of the K+and Cl–channels that facilitate solute flux for stomatal movements. A growing body of evidence now indicates that oscillations in the cytosolic free concentration of Ca2+contribute to a ‘signalling cassette’, which is integrated within these events through an unusual coupling with membrane voltage. Additional developments during the past two years point to events in membrane traffic that play complementary roles in stomatal control. Research in these areas, especially, is now adding entirely new dimensions to our understanding of guard cell signalling.
Addresses
Laboratory of Plant Physiology and Biophysics, Imperial College of Science, Technology and Medicine at Wye, Wye, Kent TN25 5AH, UK; e-mail: mblatt@wye.ac.uk
Current Opinion in Plant Biology2000, 3:196–204 1369-5266/00/$ — see front matter
© 2000 Elsevier Science Ltd. All rights reserved. Abbreviations
ABA abscisic acid
ARF ADP-ribosylation factor G-protein [Ca2+]
i concentration of cytosolic free calcium cADPR cyclic ADP–ribose
EK K+equilibrium voltage
GEF guanine-nucleotide exchange factor ICl non-inactivating Cl–current IK,in inward-rectifying K+current IK,out outward-rectifying K+current IP3 inositol-1,4,5-trisphosphate Nt-Syr1 Nicotianasyntaxin-related protein 1 PLD phospholipase D
SNARE soluble N-ethylmaleimide-sensitive factor attachment protein receptor
Introduction
Stomata are pores that form between pairs of specialised cells, called guard cells, and that are found on the epidermis of all aerial parts of most higher plants. Guard cells open and close the stoma to regulate gas exchange between the inter-cellular spaces within the plant tissue and the surrounding environment, thereby influencing two of the processes that are most important to the vegetative plant, photosynthesis and transpiration. Thus, the control of stomatal aperture has long been recognised as directly influencing vegetative yields, as well as water resource and arable land manage-ment. Guard cells have also become a focus of attention in fundamental research as a ‘model’ for higher-plant cells. Their ability to integrate environmental and endogenous signals, and their position within the leaf tissue has enabled
explorations of the signal cascades that link guard-cell membrane transport to stomatal control. Of these, the sig-nalling mechanisms evoked by the water-stress hormone abscisic acid (ABA) have received the greatest attention and, in the past four years, have yielded some of the most excit-ing new findexcit-ings in plant cell signallexcit-ing.
Stomatal movements are achieved through changes in guard-cell volume, which, in turn, are driven by the accu-mulation (during opening) and loss (during closing) of osmotically active solutes (i.e. KCl and/or other K+salts).
Flux of these inorganic solutes must take place across the plasma membrane because mature guard cells lack func-tional plasmodesmata [1,2]. K+transport is dominated by
two classes of K+channels. The first of these permits K+
movement into, but not out of, the cell and thus gives rise to an inward-rectifying current (IK,in); the second facilitates K+flux out of, but not into, the cell and therefore appears
as an outward-rectifying current (IK,out). These two
cur-rents contribute to K+ flux during stomatal opening and
closing, respectively, and can be clearly distinguished by their whole-cell, single-channel and pharmacological prop-erties [2–4]. The movement of Cl–through so-called ‘slow’
or ‘non-inactivating’ Cl– channels (I
Cl) accounts for much
of the charge balancing of the fluxes of K+, especially
dur-ing stomatal closure when it is essential that the voltage across the guard-cell plasma membrane is brought positive of the K+ equilibium voltage (E
K) for the loss of K+
through IK,out[4].
Clearly, effective control of stomatal aperture, and hence of guard-cell volume, requires the coordinated regulation of both K+ and Cl- fluxes, and implies a high degree of
integration between the signalling pathways that deter-mine the activities of IK,in, IK,outand ICl. We know now
(see Figure 1) that ABA first, evokes an inward-directed current, mediated at least in part by ICl[5,6]; second,
inac-tivates IK,in, which normally mediates K+uptake [7]; and
third, activates the current through IK,out, which together
with IClfacilitates a net loss of KCl. At least two parallel
signalling pathways that engender changes in cytosolic-free calcium concentration ([Ca2+]
i) and pH (pHi)
underpin these events [2–4]. Nonetheless, major gaps remain in our understanding of these signalling pathways. Even the function and processing of the [Ca2+]
i signal,
itself, is proving surprisingly complex. Furthermore, key developments during the past two years point to related events in membrane traffic that play equally important roles in stomatal control. Research in these areas, espe-cially, is adding entirely new dimensions to our understanding of guard-cell signalling and of plant cell biology in general.
[Ca
2+]
i
increases and Ca
2+channels
Early debates centred around the origins of the [Ca2+] i
sig-nal, whether it is internal or external to the guard cell. Considerable evidence now indicates that increases in [Ca2+]
iarise from both Ca2+entry across the plasma
mem-brane and release from intracellular stores [8–10]. Furthermore, the vacuole is certainly not the only source of Ca2+within the cell [8]. Some uncertainty remains about
the relative contributions of various pathways, which may reflect an inherent redundancy (and, hence, plasticity) within the Ca2+cascade. Nonetheless, we can expect that
the specialisation, and localisation to selected membranes within the cell, of [Ca2+]
i-signalling elements
is also important for signal encoding [11,12].
Within the guard cell, Ca2+release and Ca2+-mediated
sup-pression of IK,inare mediated by inositol-1,4,5-trisphosphate (IP3) [13,14], which is produced rapidly in response to ABA
[15,16]. This lipid metabolite probably interacts with IP3
-sen-sitive Ca2+ channels within the cell to potentiate a rise in
[Ca2+]
i [17]. Several studies have, however, described a
sec-ond class of endomembrane Ca2+-release channels in higher
plants [17,18]. These channels are sensitive to the alkaloid ryanodine and are activated by binding cyclic ADP–ribose (cADPR), a metabolite of nicotinamide adenine dinucleotide.
A statistical analysis of cADPR-injected mesophyll had impli-cated a role for this agonist in r29Aand kin2gene expression evoked by ABA [19]. More recently, Leckie et al. [20••] have
provided direct evidence of cADPR-sensitive, Ca2+
-perme-able channels in guard-cell vacuoles and suggested that they function as a Ca2+-release pathway during ABA stimulation.
In support of this hypothesis, they observed increases in [Ca2+]
ifollowing injection of cADPR into guard cells, and a
slowing of stomatal closure in response to ABA when guard cells were preloaded with the cADPR antagonist 8NH2
-cADPR. Grabov and Blatt [21•] offer parallel evidence that
voltage-evoked increases in [Ca2+]
iare suppressed by
ryan-odine in intact guard cells. The latter study is of particular interest because it offers the first quantitative analysis of the [Ca2+]
idependence of IK,in. Grabov and Blatt [22••] had
pre-viously found evidence of a hyperpolarisation-activated Ca2+
channel at the plasma membranne, implicated by the sensi-tivity of [Ca2+]
i increases to the membrane voltage. In the
more recent study [21•], they made use of changes in clamp
voltage to elicit changes in [Ca2+]
i, monitoring IK,in under
voltage clamp. They found a high (‘switch-like’) sensitivity of the current to [Ca2+]
i, with an apparent K1/2(~330µM) only
marginally above the mean resting [Ca2+]
i. Whether ABA
affects the [Ca2+]
idependence of IK,inis not known;
howev-er, ABA does strongly influence the voltage sensitivity of Figure 1
ABA leads to a concerted modulation (⊕, activation; O, inactivation) of three plasma membrane ion channels: the two dominant K+ currents (a) IK,in, (b) IK,out, and (c) the slow-activating anion current ICl(and, probably also (d)the H+-ATPase). ABA binds to an as yet unidentified receptor (X), but that is postulated to be on the plasma membrane. Binding may activate a G-protein (Gα) and trigger activation of phospholipase C (PLC)-mediated hydrolysis of phosphoinositol bisphosphate to IP3, thereby releasing Ca2+ from intracellular stores and the vacuole. (e) ABA also affects Ca2+entry across the plasma membrane and its interaction with intracellular Ca2+-release pathways. High-gain switch-like Ca2+-induced and (f) cADPR-mediated Ca2+release from intracellular stores affects IK,in, ICland the H+-ATPase. Parallel but independent increases in pHiact on IK,in, IK,out, ICl, and deplete the substrate for the H+-ATPase. Additional targets and interactions with the abi1 protein
phosphatase (abi1) and other protein kinase (PK)/protein phosphatase (PP) activities (P*) are discussed in several recent reviews [8,10,83].
rise and its return to resting concentrations [22••]. So, we can
anticipate a compound action of ABA both on Ca2+ entry
across the plasma membrane and on its release from cADPR-dependent or other Ca2+stores.
The spatial distribution of internal Ca2+-release pathways
is also likely to affect the kinetics of [Ca2+]
ichanges and
Ca2+-signal encoding. The vacuole is an obvious source of
Ca2+ and the tonoplast contains slow vacuolar (SV)-type
channels that may be related to animal ryanodine-receptor Ca2+channels [23•], and contribute to Ca2+ release [24].
Nonetheless, Ca2+ channels have also been identified in
the endoplasmic reticulum (ER) of mechanosensitive organs in Bryonia [25] and probably the ER of Lepidium roots [26]. In Brassica florets, Ca2+release appears to be
triggered by IP3in vesicular compartments that are distinct
from the vacuole [27]. Furthermore, in the giant alga Chara, the action potential (which is initiated by Ca2+
-mediated activation of Cl-channels [28,29•,30]) draws on
IP3-dependent Ca2+release from a non-vacuolar
compart-ment close to the plasma membrane. In a particularly elegant study, Plieth et al. [31••] preloaded Chara with
Mn2+, targeting either all intracellular stores (by incubation
in Mn2+medium) or only the central vacuole (by injection
with Mn2+). They found that Mn2+quenches the
fluores-cence of the Ca2+-sensitive dye Fura2 and, because it also
permeates many Ca2+ channels, it provides an assay for
divalent release. When they evoked action potentials, Plieth et al. [31••] observed a quenching of Fura2
fluores-cence in cells that were uniformly loaded with Mn2+, but
not in cells in which Mn2+was loaded only into the
vac-uole. Whether spatial differences in Ca2+ release
underpins [Ca2+]
i signalling in guard cells has yet to be
determined, but there is certainly evidence of local differ-ences in [Ca2+]
idynamics between the peripheral cytosol
and regions of the cell close to the nucleus [22••]. To
sum-marise, both the pharmacological and kinetic characteristics, as well as the spatial distribution of Ca2+
-release pathways within the guard cell, can be expected to play important roles in defining the nature of the [Ca2+] i
response to ABA.
[Ca
2+]
i
oscillations
Ca2+mediates a diversity of responses, even within a
sin-gle cell. The issue of how a sinsin-gle second messenger could give rise to such varied responses was resolved only when it was recognised that the frequency and location of a sequence of repetitive increases in [Ca2+]
iwithin the cell
might determine the nature of the response(s) [12,32,33]. Frequency modulation of [Ca2+]
iincreases or ‘spikes’ has
been shown to give rise to ‘Ca2+ signatures’ that encode
specific responses, including gene expression [34,35] and calmodulin-dependent protein kinase activity [36•]. In
ani-mals, [Ca2+]
i oscillations initiate Ca2+ entry across the
plasma membrane; local [Ca2+]
ielevation then triggers
fur-ther Ca2+ release from intracellular stores (i.e.
Ca2+-induced Ca2+ release [CICR]) before the Ca2+ is
The result is repetitive [Ca2+]
ispikes, each with a duration
of a few seconds or less.
Interest in [Ca2+]
isignalling in plants has been heightened
by observations that [Ca2+]
i may oscillate in response to
external stimuli [37–40]. In guard cells, however, these oscillations often take place over periods of more than 10 min, with discrete [Ca2+]
imaxima lasting for 2–4 min or
more when evoked by a rise in the extracellular Ca2+
con-centration or CO2[40,41]. A similar oscillation pattern has
now been observed in response to ABA. Staxen et al. [42•]
reported [Ca2+]
i increases from approximately 200 nM at
rest to 400–600 nM within 15–30 min of exposure to 10 nM ABA. Significantly, treatments with higher concentrations of ABA that resulted in a pronounced decrease in stomatal aperture also produced a reduced amplitude in the [Ca2+] i
oscillations and a longer duration of the [Ca2+]
ielevation. A
role for IP3-mediated Ca2+release was suggested because
the ABA-evoked oscillations were partially suppressed by treatment with the phospholipase C antagonist U73122.
This pattern of slow and irregular oscillations in [Ca2+] i
raises questions about the origins and role of this signal in the guard cell. One explanation relates [Ca2+]
ioscillations
to the voltage status of the cell. Guard cells, like many higher-plant cells, have two states of membrane voltage: one state close to EK, and the other largely K+-insensitive
and typified by voltages well negative of EK [43,44].
Transitions between these states take place in response to stimuli, including ABA, that can give rise to oscillations in voltage with periods of between 10 s and many minutes [43,45]. Because membrane hyperpolarisation also evokes a Ca2+influx and a consequent rise in [Ca2+]
i, it is most
likely that the [Ca2+]
i oscillations mirror an underlying
oscillation in plasma membrane voltage [22••]. These
char-acteristics — and the effect of ABA in altering the voltage threshold for the [Ca2+]
i rise [22••] — suggest a role for
[Ca2+]
ielevation in a feedback mechanism that ‘tunes’ the
[Ca2+]
i-dependent currents IK,inand IClto the prevailing
transport status of the guard cell. Thus, [Ca2+]
i feedback
creates a response ‘cassette’ for osmotic balance (Figure 2), switching the membrane between states in which net uptake and net loss of K+and Cl–take place [8,44].
Membrane traffic and cell volume
The activation of Ca2+channels may also play a role in
events one step removed from the regulation of K+and Cl–
of these issues in Charamore than two decades ago. Yet, despite their implied function in stomatal movements, the dynamics of membrane trafficking within the guard cell were largely ignored until recently. Two recent develop-ments have no placed membrane trafficking in the limelight of stomatal physiology and ABA signalling.
In the first of these, Homann [50•] and Kubitscheck et al.
[51••] used in vivo capacitance recording and
membrane-surface labelling with styryl dyes to quantify membrane exocytosis and endocytosis in guard-cell protoplasts during osmotically driven volume changes. Capacitance recording makes use of sine-wave retardation to determine mem-brane capacitance [52–54], which is directly proportional to the membrane surface area. An increase in capacitance represents the sum of all exocytotic events less the sum of all endocytotic events. Endocytotic events can subse-quently be isolated by simultaneously monitoring internalisation of fluorescent styryl dyes, such as FM1-43 [55]. Homann [50•] found that the membrane surface area
of Viciaguard-cell protoplasts increased under hypo-osmot-ic conditions and decreased after hypertonhypo-osmot-ic treatment, and that the rate of change in each case was graded in response to the magnitude of the osmotic potential differ-ence. Kubitscheck et al. [51••] subsequently combined
these techniques with FM1-43 dye measurements. Their results demonstrate a rapid internalisation of membrane during hyperosmotic stimulation that is quantitatively con-sistent with the decrease in plasma membrane surface area (and also capacitance). Intriguingly, the dye also accumu-lated gradually within the cell in a ring 1–2µm below the plasma membrane, even in the absence of an osmotic step. This steady internalisation suggests that constitutive endocytosis continues under constant osmotic pressure, resulting in the accumulation of a pool of sub-plasma membrane vesicles. Whether these vesicles are available for re-incorporation into the plasma membrane remains to be determined. The role of ABA and of Ca2+ in these
events can also be expected to draw attention in the near future. So far, all of the evidence indicates that Ca2+does
not have a direct impact on the membrane trafficking that is evoked by osmotic gradients [50•]. It may be that
osmot-ic potential- and ABA-sensitive traffosmot-icking controls overlap only in part. Whether or not this is the case, experimental results point to a highly dynamic process of membrane cycling that is responsive to membrane tension.
Figure 2
Cytoplasm Outside
Ca2+
Ca2+ Ca2+ Ca2+
Ca2+
K+
Cl−
+ ∆ψ
cADPR IP3
Intracellular compartment
(a)
Cytoplasm Outside
Ca2+
Ca2+ Ca2+ Ca2+
Ca2+
K+
Cl−
+ ∆ψ
cADPR IP3
Intracellular compartment
(b)
Cytoplasm Outside
Ca2+
Ca2+ Ca2+ Ca2+
Ca2+
K+
Cl−
+ ∆ψ
Intracellular compartment
(c)
Cytoplasm Outside
Ca2+
Ca2+ Ca2+ Ca2+
Ca2+
K+
Cl−
+ ∆ψ
Intracellular compartment
(d)
Current Opinion in Plant Biology
Ca2+ channel
Cl– channel
K+ channel
Figure 2 Legend
The voltage–[Ca2+]
iresponse ‘cassette’ cycle. (a) Negative membrane voltage (downward arrow indicates amplitude) triggers Ca2+influx across the plasma membrane that (b) stimulates intracellular Ca2+ release, activates Cl–channels for Cl–efflux and inactivates I
K,in. (c) The rise in [Ca2+]
i, bias to Cl–efflux and additional changes in membrane conductance (not shown) lead to membrane depolarisation, inactivate the Ca2+influx and engage Ca2+extrusion processes. (d) Finally, with [Ca2+]
The second development has come from the identifica-tion of the Nicotiana syntaxin homologue Nt-Syr1. Syntaxins belong to a group of integral membrane pro-teins that form the core of the molecular machinery for vesicle trafficking and membrane fusion (see below). Surprisingly, Leyman et al. [56••] isolated the Nt-Syr1
gene by expression cloning in a heterologous screen designed to identify an ABA receptor. Their studies demonstrate not only that ABA enhances Nt-Syr1 tran-script and Nt-Syr1 protein levels in the plant but also that disrupting Nt-Syr1 function, with either the Clostridial neurotoxin BotN/C (an endopeptidase that specifically cleaves the syntaxin) or by loading guard cells with the soluble (carboxy-truncated) portion of Nt-Syr1, prevents both K+and Cl–channel responses to
ABA in vivo. These results, and observations of a high molecular weight band recognised by Nt-Syr1 antibod-ies, suggest that Nt-Syr1 functions in a complex that is competitively blocked through substitution with the car-boxy-truncated protein. They also indicate that Nt-Syr1 functions within or very close to the early steps of the ABA signal cascade, either as a scaffolding protein, a sec-ond messenger or a modulator of the activity/availability of one or more signalling elements.
Further evidence of membrane trafficking in
stomatal control?
Two additional lines of evidence hint at roles for mem-brane trafficking in guard-cell volume control and ABA signalling. First, in screening for ABA-hypersensitive mutants of Arabidopsis, Cutler et al. [57] identified Era1, the gene that encodes a protein farnesyl-transferase. Farnesyl- and geranylgeranyl-transferases covalently add lipid moieties to small GTPases, anchoring the latter at the membrane surface [58] to target GTPase activity in a vari-ety of cellular functions. Although the substrate(s) for Era1 is not known, Rac- and Rho-type GTPases, which are important for cytoskeletal function [59], are commonly far-nesylated. In fact, a Rho-like GTPase, Rop1, that is localised to the apex of Pisumpollen tubes [60] has been implicated in microfilament-based intracellular transport of vesicles. Intriguingly, actin organisation has also been suggested to regulate guard-cell ion channels and stomatal movements, although the function of actin in this case has been interpreted in the context of control by mechanical stress rather than membrane trafficking [61–63].
The Era1 farnesyl-transferase also contributes to ABA sig-nalling. Pei et al. [64•] have reported an enhanced
(b) SNARE as a receptor/signal intermediate coupling element
ABA
Net endocytosis Net endocytosis
Ion channel control
Ion channel control Ca2+
pHi Ca2+
pHi (a) SNARE as a scaffolding/actuator element
Current Opinion in Plant Biology
Two models for the dual functioning of Nt-Syr1 (and other SNARE proteins) in controlling ion channels and vesicle trafficking. Soluble ABA (orange) binding to its receptor (blue) alters both net endocytosis and the permeation of ions (yellow) through ion channels (red).
sensitivity of guard-cell Cl– channels to ABA in the era1
mutant and in wild-type Arabidopsis plants after treatment with broad-range farnesyl-transferase antagonists. The era1 phenotype is dominant and persists throughout the life cycle of the plant. So, although these results do not dis-tinguish between possible roles for Era1 as an ABA-signalling element per se and as a secondary element necessary to maintain the signalling machinery, they do implicate a role for small GTPases and potentially for membrane trafficking in guard-cell function.
Second, Jacob et al. [65•] have recently reported a
tran-sient rise in the phospholipase D (PLD) activity of guard-cell protoplasts following stimulation with ABA. They also observed a reduction in the activity of IK,in
that complemented the action of the cADPR antagonist nicotinamide when the protoplasts were treated with the PLD by-product phosphatidic acid. It is worth noting that both PLD and phosphatidic acid have been impli-cated in secretion by the barley aleurone [66]. Phosphatidic acid contributes to the regulation of vesicle trafficking at the Golgi apparatus [67], and PLD activity has been associated with the functioning of ARF- and Rho-GTPases in the secretory processes of mammals and yeast [68,69]. Once again, the data suggest a link between membrane trafficking and ABA.
Coordinating membrane traffic and ion
channel control
How might membrane trafficking contribute to ion-chan-nel control and ABA signalling? Membrane cycling and fusion between the membranes of eukaryotic cells are facilitated by a family of SNARE (i.e. soluble N -ethyl-maleimide-sensitive factor attachment protein receptor) proteins that are associated with each membrane [70–72]. During synaptic transmission, which depends on vesicle fusion and release of a neurotransmitter, the target (t-SNARE) proteins syntaxin and SNAP-25 form a stable ternary complex with the vesicle (v-SNARE) protein synaptobrevin that serves to bring the two membrane bilayers into close proximity for fusion [73]. In yeast and plants, SNARE proteins are thought to be essential for the related functions of interorganelle transport, growth and cell division [72,74]. Thus, SNAREs have been recognised as contributing to response mechanisms and not as compo-nents of signal cascades per se.
Even so, SNAREs are probably important for signal transduction, its plasticity and adaptation. SNAREs may contribute to ion-transport regulation simply by mediat-ing the selective addition and removal of transport proteins from the membrane. These events can take place with remarkable rapidity. A recent study of the
Arabidopsis GNOM gene, which encodes a GTP–GDP
exchange factor (GEF) for an ADP-ribosylation factor G-protein (ARF) GTPase, is a case in point. Steinmann et al. [75••] report that treatments with the ARF–GEF
antagonist brefeldin A result in dramatic alterations in
protein localisation within 15–30 min, including a loss of the polar distribution of the putative auxin efflux carrier protein Pin1. Auxin efflux, itself, is altered within 10 min of brefeldin A treatments [76], suggesting that substan-tial changes to the make-up of the membrane transport proteins take place within the same time period. The data certainly highlight the dynamic flux of membrane and protein within the plant cell.
The role(s) carried out by SNAREs may prove to be much more subtle still. In neurons, SNAREs, including syntax-ins, bind to and regulate Ca2+channels [77,78] as well as
CFTR Cl-channels [79]. Syntaxins interact with proteins
that may not be related to secretion processes directly, including the orphan G-protein-coupled receptor CIRL [80] and tomosyn, a microfilament-associated protein [81]. Furthermore, SNARE proteins have been associated with responses to membrane tension and osmotic stress [82]. So, one possibility (Figure 3) is that SNARE proteins such as Nt-Syr1 [56••] contribute to the scaffolding of an
ABA-receptor complex and, therefore, serve dual roles by marrying the functions of a signalling element and of the machinery for membrane trafficking.
Conclusions
Research into the cellular and molecular mechanisms of stomatal control has begun to take on new dimensions in relating signalling pathways with response ‘cassettes’ that comprise several ion transporters and their control mecha-nisms. Understanding how these cassettes are assembled will present a major challenge in the coming years. Defining the interactions within these cassettes is already providing a clearer picture of the functions of Ca2+as a
sec-ond messenger. At the same time, attention to the related processes of membrane dynamics and trafficking promises to add to our understanding of both cellular transport and volume control, as well as to the guard-cell system as a higher-plant cell model.
Update
Following up the evidence of a hyperpolarisation-activated rise in [Ca2+]
i, Hamilton et al. [84••] have now identified a
small-conductance Ca2+channel at the plasma membrane
by patch-clamp of Viciaguard-cell protoplasts. The chan-nel shows characteristics that are entirely consistent with those expected from the previous studies, including a dependence on negative voltage and block by Gd3+and
La3+[21•,22••]. Furthermore, ABA evokes a large increase
in channel opening, even in isolated membrane patches. These results suggest that the ABA receptor is physically close to the Ca2+channel and localised to the inner surface
of the plasma membrane.
Acknowledgements
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