Phosphatidylinositol 5-Kinase Stimulates Apical Biosynthetic Delivery via an Arp2/3-Dependent ...

21 Pages·2006·3.74 MB·English

JBC Papers in Press. Published on April 6, 2006 as Manuscript M601239200
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M601239200

Phosphatidylinositol 5-Kinase Stimulates Apical Biosynthetic
Delivery via an Arp2/3-Dependent Mechanism
Christopher J. Guerriero, Kelly M. Weixel, Jennifer R. Bruns, and Ora A. Weisz*
From the laboratory of Epithelial Cell Biology, Renal-Electrolyte Division, University of
Pittsburgh, Pittsburgh, PA 15261
Running title: Actin-comet mediated apical transport
Address correspondence to: Ora A. Weisz Renal-Electrolyte Division University of Pittsburgh
3550 Terrace St. Pittsburgh, PA 15261 Tel: [phone] Fax: [phone] E-Mail: [email protected]

The mechanisms by which polarized epithelial newly synthesized proteins and lipids to
cells target distinct carriers enriched in newly differentiated apical and basolateral plasma
synthesized proteins to the apical or basolateral membrane domains. Tight junctions between the
D
o
membrane remain largely unknown. Here we cells prevent the diffusion of surface proteins w
n
investigated the effect of phosphatidylinositol between these domains, but polarity is established lo
a
d
metabolism and modulation of the actin and maintained largely by the selective delivery ed
cytoskeleton, two regulatory mechanisms that and recycling of proteins to their appropriate site fro
m
have individually been suggested to function in of function. In polarized renal cells, it is thought h
ttp
biosynthetic traffic, on polarized traffic in that newly synthesized proteins are sorted initially ://w
MDCK cells. Overexpression of at the trans-Golgi network (TGN) into distinct w
w
phosphatidylinositol 5-kinase (PI5K) increased carriers destined for the apical or basolateral .jb
c
actin comet frequency in MDCK cells and domain (1,2). Transport of some proteins to their .o
rg
concomitantly stimulated TGN-to-apical ultimate destination may be indirect and include b/
y
membrane delivery of the raft-associated passage through endosomal compartments or the g
u
e
protein influenza HA, but did not affect opposite surface domain (2). st o
delivery of a non-raft associated apical protein The sorting of individual proteins to the n A
p
ocor mae tb afosromlaatetrioanl mbya rpkhearr. mMaocdoluolgaitcio mn eoafn sa,c tbiny asipgincaall -maneddi abteads.o latBeraaslo lcaetellr als urfsaocret indgo masiingsn ailss ril 2, 2
0
overexpression of the TGN-localized inositol generally reside in the cytoplasmically-disposed 1
9
polyphosphate 5-phosphatase Ocrl, or by regions of proteins whereas glycan-, lipid-, and
blockade of Arp2/3 function had parallel effects peptide-dependent signals have been identified
on the rate of apical delivery of HA. Moreover, that reside in the lumenal, membrane-associated,
HA released from a TGN block was colocalized or cytoplasmic regions of distinct apically-targeted
in transport carriers in association with PI5K proteins (2). Little is known about how the sorting
and actin comets. Inhibition of Arp2/3 function machinery recognizes these diverse signals;
in combination with microtubule however a current model suggests that preferential
depolymerization led to a virtual block in HA incorporation of a subset of apical proteins,
delivery, suggesting synergistic coordination of including those with glycosphingolipid anchors or
these cytoskeletal assemblies in membrane sorting signals within their transmembrane
transport. Our results suggest a previously domains, into glycolipid-enriched microdomains
unidentified role for actin comet-mediated (lipid rafts) is important for their polarized
propulsion in the biosynthetic delivery of a delivery (1). Interestingly, apical delivery of raft
subset of apical proteins. vs. non-raft proteins may involve distinct transport
carriers that are independently regulated (3,4).
The maintenance of polarized cell function There is increasing evidence for a role of
requires continuous active sorting and delivery of phosphatidylinositols in the regulation of
1
Copyright 2006 by The American Society for Biochemistry and Molecular Biology, Inc.
biosynthetic membrane traffic (5). The Golgi protein. MDCK cells overexpressing PI5K had
contains a sizable pool of phosphatidylinositol 4- markedly increased numbers of actin comets, and
phosphate (PI4P) and harbors two PI4P- HA could be visualized at the tips of these
synthesizing enzymes (PI4KIIIβ and PI4KIIα; (6- structures. Disruption of actin comet formation
9)). Our lab has previously demonstrated that together with microtubule depolymerization led to
overexpression of PI4KIIIβ inhibits the rate of a nearly total blockade in apical delivery,
apical membrane delivery of the raft-associated suggesting a concerted role of these distinct
protein influenza HA, whereas expression of a cytoskeletal assemblies for efficient and polarized
kinase-deficient mutant stimulates delivery (8). apical transport. Our data suggest that actin comets
However, it is conceivable that these effects are contribute selectively to the polarized delivery of
due to a downstream metabolite of PI4P, such as apically-destined transport containers enriched in
phosphatidylinositol 4,5-bisphosphate (PIP ). lipid raft-associated cargo.
2
Indeed, although there is only a small amount of
this lipid that can be visualized in the Golgi Experimental Procedures
complex (10), PIP is readily generated on isolated
2
Golgi membranes incubated with DNA and replication-defective recombinant
phosphatidylinositol 4-phosphate 5 kinases adenoviruses –The alpha isoform of murine
[PI5Ks; (11)] and several possible functions for phosphatidylinositol 5-kinase (PI5K) cloned into D
o
PIP in biosynthetic membrane traffic have been the pAdtet vector was provided by Andreas w
2 n
postulated (5,12,13). Additionally, the presence of Jeromin (Baylor University). Constructs encoding loa
d
Ocrl, a TGN-localized PIP 5’-phosphatase that is myc-tagged W and WA domains of WAVE1 were ed
defective or absent 2 in patients with generous gifts of Dr. Dorothy Schafer and Dr. fro
m
oculocerebrorenal syndrome of Lowe, lends James Casanova (University of Virginia). h
ttp
further support to a role for PIP2 metabolism in Fluorescent protein-tagged actin constructs were ://w
that compartment (14,15). provided by Dr. Ronald Montelaro (University of w
w
Increases in PIP2 levels upon expression Pittsburgh). The generation and purification of .jbc
of PI5Ks leads to production of rapidly nucleating replication-defective recombinant adenoviruses .org
branches of actin filaments, termed actin comets, (AVs) encoding tetracycline-repressible influenza b/
y
which are capable of propelling transport vesicles hemagglutinin (HA; Japan serotype), vesicular gu
e
through the cytoplasm (16). Neuronal Wiskott- stomatitis virus G protein (VSV-G), and a control st o
Aldrich syndrome protein (N-WASP), a potent virus (encoding the influenza Rostock M2 coding n A
p
sctoimmupllaetxo, r iso f acatcivtiant edn ucblye atPioIPn 2 vaina d thtera nAsdrupc2e/3s sperqevuieonucsel y idne stchreib erde v(e8r,s1e9 ).o rAieVnsta teinocno) dihnags PbI5eKen, ril 2, 2
0
elevated PIP levels into an increased frequency of and human Ocrl (both wild type and the 1
2 9
actin comets (17). N-WASP and Arp2/3 are phosphatase-deficient mutant R483G; constructs
localized to the Golgi complex (among other provided by Dr. Robert Nussbaum, N.I.H.) were
sites), and a link between actin comets and generated using similar methods. AV encoding
biosynthetic traffic has been previously suggested p75NTR was provided by Dr. Enrique Rodriguez-
(18). However, the effects of manipulating PI5K Boulan (Weill Medical College) with permission
or Arp2/3 activity on the fidelity or rate of from Dr. Moses Chao, and AV-W and WA were
biosynthetic transport have not been explored. kind gifts of Dr. James Casanova (University of
Here we have examined the effect of Virginia).
increasing PIP synthesis on polarized biosynthetic
2
traffic in Madin-Darby canine kidney (MDCK) Cell lines and adenoviral infection- MDCK type II
cells. Expression of PI5K selectively stimulated cells stably expressing tetracycline transactivator
the TGN-to-apical delivery of a raft-associated were cultured in Modified Eagles’s medium
protein without affecting the overall polarity of (Sigma) supplemented with 10% fetal bovine
delivery. Moreover, the effect of PIP appears to serum. For measurements of intra-Golgi transport,
2
involve an Arp2/3-dependent pathway, as a kinetics of surface delivery, and surface polarity,
dominant-negative inhibitor of Arp2/3 function cells were seeded at superconfluence in 12-mm
selectively inhibited biosynthetic transport of this transwells (0.4-µm pore; Costar, Cambridge, MA)
2
for 2–4 days prior to infection with recombinant measure TGN to surface delivery, radiolabeled
AVs at the following multiplicity of infection cells were chased at 19oC for 2 h unless otherwise
(m.o.i.; control AV, AV-PI5K, AV-W, AV-WA indicated in order to stage newly synthesized
m.o.i. 100-250; AV-HA, AV-p75, AV-VSV G, membrane proteins in the TGN. The cells were
m.o.i. 25-50) as described in (19). Experiments then rapidly warmed to 32oC or 37oC as indicated.
were performed the following day. Apical delivery of HA was measured by
trypsinization as described in (21). Basolateral
Indirect Immunofluorescence—MDCK cells delivery of VSV G was quantitated using domain
grown on transwell filters or coverslips were selective biotinylation as described in (22). To
infected with AVs at the indicated m.o.i. The quantitate surface delivery of p75NTR, cells were
following day, cells were rinsed once with PBS, radiolabeled on 100 µl drops of sulfate-free
fixed in 3.7% formaldehyde, rinsed with PBS medium containing 50 µCi [35S]-sulfate at 19oC
containing 10 mM glycine (PBS-G), then and the rate of surface delivery was assessed using
permeabilized with 0.5% Triton X-100 in PBS-G domain selective biotinylation upon subsequent
for 3 min at room temperature. After washing, warmup to 37oC (23). Immunoprecipitation of HA,
nonspecific binding sites were blocked by VSV-G, or p75 was performed using supernatants
incubation for 5 min in PBS-G containing 0.25% from cultured hybridomas (Fc125 from Dr.
w/v ovalbumin. Coverslips were incubated for 30 Thomas Braciale, University of Virginia; 8G5 D
min with monoclonal anti-HA tag antibody (1:500 from Dr. Douglas Lyles, Wake Forest University ow
n
dilution; Covance) followed by washing in (24); and MA 20.1 from Dr. Enrique Rodriguez- loa
d
blocking buffer. Cells were then incubated for 30 Boulan, Weill Medical College, respectively). ed
min with secondary antibodies Alexa Fluor goat fro
m
anti-mouse 488 (1:500; Invitrogen, Carlsbad, CA). Visualization and quantitation of actin comets- h
Rhodamine phalloidin (1:80; Invitrogen) was MDCK cells (2 x 105) were seeded on Bioptech ttp://w
included in this step where indicated. After 0.17 mm ∆T dishes (Bioptech Inc., Butler, PA). w
w
extensive washing, coverslips or filters were The following day cells were infected with AVs .jb
c
mounted onto slides. Filter-grown MDCK cells (m.o.i. 100-250) encoding the indicated proteins, .org
were fixed with formaldehyde using a pH-shift and incubated overnight with 0.25 ng/ml b/
y
protocol and processed as described in (20). doxycycline (DOX) to partially suppress protein gu
e
Images were captured using a Leica TCS-SL expression. The following morning DOX was st o
confocal microscope equipped with argon and removed by thorough washing. Cells were then n A
IgLre)e. n Iamnda greeds hweleiurem nteaoknen l aswerist h( Leai ca1, 0D0eXa rfi(e1l.d4, pYrFesPs-uarcet-inin jaencdte dw ewreit hr ectuDrnNeAd teon ccoudltiunrge GfoFrP 5- ohr. pril 2, 2
numerical aperture) plan apochromat oil objective. Following that time the cells were imaged on an 01
9
TIFF images were processed using Adobe Olympus IX81 microscope using a 100X Olympus
Photoshop (Adobe, San Jose, CA). UPlanApo objective (numerical aperture 1.35).
Random fields containing fluorescent-actin-
Intracellular transport and cell surface delivery expressing cells were imaged every 2 sec for 4
assays- AV-infected filter-grown MDCK cells min. Data were analyzed using acquisition
were starved for 30 min and radiolabeled for 5-10 software (Slidebook) to determine the percentage
min (endo H kinetics) or 15-20 min (TGN-to- of cells with comets. Stable cells lines expressing
surface delivery) with 1mCi/ml Tran-[35S]-Label® fluorescent protein-tagged actin were generated
(MP Biomedicals, Irvine, CA). To measure endo using Lipofectamine 2000 and mixed populations
H kinetics, cells were chased in bicarbonate-free isolated by selection in G418. These cells were
MEM for the indicated periods, solubilized, and infected with AV-PI5K where indicated and used
HA immunoprecipitated as previously described in for the quantification of actin comets in cells
(19). After collection of antibody-antigen treated with phorbol-myristate acetate (PMA; 5
complexes, samples were eluted, divided in half, µg/ml), cytochalasin D (25 µg/ml), n-butanol (1%
and mock-treated or treated overnight with v/v), and t-butanol (1% v/v).
endoglycosidase H (endo H) prior to
electrophoresis on 10% SDS-PAGE gels. To
3
Visualization of cargo associated with actin mutant of Ocrl) on the delivery of HA and p75.
comets- MDCK cells (3 x 105) were seeded onto Because overexpression of both wild-type and
coverslips in 12-well dishes. The following day mutant Ocrl slowed intra-Golgi transport as
the cells were co-infected with AVs encoding HA assessed by monitoring endo H kinetics, we used
and either PI5K or control AV. After 8 h at 37oC, [35S]-sulfate to selectively radiolabel proteins in
the cells were incubated at 19oC for 2.5 h to stage the TGN. As shown in Fig. 1E, expression of wild-
HA in the TGN. Dishes were then warmed to 37oC type Ocrl inhibited TGN-to-apical delivery of HA
for 0 or 30 min before fixation and processing for whereas mutant Ocrl had no effect. Consistent
indirect immunofluorescence to detect HA, PI5K, with the selective effect of PI5K on HA delivery,
and actin. neither wild-type nor mutant Ocrl affected the
RESULTS kinetics of p75 delivery (Fig. 1F).

PI5K selectively stimulates biosynthetic delivery PI5K stimulates the formation of actin comets in
of an apical raft-associated protein- We MDCK cells- Because PI5K-mediated actin
examined the effect of AV-mediated comets have been implicated in the transport of
overexpression of murine PI5Kα on biosynthetic raft-associated proteins (18), we asked whether
traffic in polarized MDCK cells. Overexpression actin comet-mediated propulsion of HA transport
of PI5K increased cellular levels of PIP2 by 2-3 carriers could be responsible for the stimulated Do
fold (not shown) but did not alter intra-Golgi surface delivery we observed. First, we sought to w
n
kinetics based on the quantitation of the rate of determine the relationship between PI5K and actin loa
d
acquisition of resistance to endoglycosidase H in fixed cells. MDCK cells were infected with AV ed
(endo H) digestion of influenza hemagglutinin expressing PI5K, then fixed and processed for fro
m
(HA; Fig. 1A). To examine the effect of PI5K on indirect immunofluorescence 16 h later. Actin http
the kinetics of post-Golgi transport, we staged filaments were visualized using rhodamine ://w
newly-synthesized radiolabeled HA in the trans- phalloidin and the HA epitope tag on PI5K was w
w
Golgi network (TGN) at 19oC, and using a cell detected using a monoclonal mouse antibody. .jb
c
surface trypsinization assay measured the rate of PI5K colocalized extensively with actin filaments .org
its delivery to the apical cell surface upon in confocal sections of MDCK cells (Fig. 2A). b/
y
warming to 37oC. Roughly 50% of the total HA This is consistent with previous reports that PI5K gu
e
expressed in control cells reached the apical is found in actin-rich fractions isolated from st o
membrane within 1 h (Fig. 1B). In contrast, HA thrombin-activated platelets (25). Next, we used a n A
p
doevleirveexrpyr eswsiansg PIm5aKr k(e~d6l7y% astti 1m hu)la. t ed in cells lsitvime uclaetlel s atphper ofaocrhm attoio nd eotefr macintien wcohmetehtesr. APIc5tiKn ril 2, 2
0
We next examined the effect of PI5K on comets have not previously been reported in 19
the TGN-to-surface transport kinetics of p75, an MDCK cells. MDCK cells were seeded on glass
apical marker not known to associate with lipid coverslips, infected with adenovirus (AV)
rafts in MDCK cells (Fig. 1C), as well as on the encoding PI5K or control AV, and then incubated
basolateral marker vesicular stomatitis virus G in the presence of 0.25ng/ml doxycycline (DOX)
protein (VSV G; Fig. 1D). PI5K had no effect on to suppress PI5K expression. The following day,
the biosynthetic delivery of either of these the DOX was washed out and cells were
proteins. Thus, the stimulatory effect of PI5K on microinjected with cDNA encoding GFP-actin.
surface delivery appears to be selective for raft- Random fields were imaged 5 h later for 4 min
associated proteins. intervals using an Olympus IX81 equipped with a
100X Olympus UPlanApo objective. Actin comets
Overexpression of Ocrl selectively inhibits were defined as rapidly moving bursts of actin
biosynthetic delivery of influenza HA- To followed by a fading tail of presumably
examine whether stimulation of PIP synthesis in depolymerizing actin. Comets generated in MDCK
2
the TGN was responsible for the stimulatory effect cells were distinct but generally smaller than
of PI5K, we compared the effect of overexpressing comets described by others using different cell
the TGN-localized PIP 5’-phosphatase Ocrl (or as lines, which have been reported to be up to 5 µm
2
a control, expressing a phosphatase-deficient in length (18,26,27). Comets were observed in 7%
4
of control cells and in 27% of the cells expression of these constructs (Figs. 4C and D),
overexpressing PI5K (Table 1; Fig. 2B, and although we did notice in cells expressing very
supplementary movie). high levels of WA that the Golgi appeared to be
Additionally, we were able to increase the somewhat dispersed towards the edges of the cells.
frequency of actin comet formation in control cells Interestingly, whereas WA was diffusely
by addition of the PKC activator phorbol-myristate expressed throughout the cytoplasm of polarized
acetate (PMA), which has been previously MDCK cells as expected, PI5K was concentrated
demonstrated to stimulate actin comets in vivo near the apical membrane, a region that contains
[Table 1; (16)]. PMA also stimulated the rate of the supranuclear Golgi complex and the actin-rich
apical delivery of HA to an extent comparable to terminal web of these cells (Fig. 4C).
that observed upon overexpression of PI5K (Fig. We next assessed the impact of W or WA
3). Conversely, addition of n-butanol, which domain expression on early transport of HA
disrupts phospholipase D-mediated synthesis of through the biosynthetic pathway by monitoring
the PI5K activator phosphatidic acid (PA), the kinetics of HA acquisition of endo H
profoundly inhibited the formation of PI5K- resistance. Expression of WA or W had no
stimulated actin comets (Table 1) and also blocked discernable effects on the kinetics of HA traffic
delivery of TGN-staged HA to the apical surface through the early Golgi (data not shown). Next, we
(Fig. 3). N-butanol-mediated inhibition of PA examined the effects of these domains on HA
D
o
synthesis has previously been implicated in the delivery from the TGN to the plasma membrane. w
n
release of secretory vesicles from the TGN of Expression of WA inhibited kinetics of HA lo
a
d
endocrine cells (28). In contrast, t-butanol, which delivery to the apical cell surface by ed
does not affect PA synthesis, had no effect on approximately ~50-75% relative to control over a fro
m
comet formation or HA delivery (Fig. 3). 60 min chase period (Figs. 5A and B). Expression h
ttp
of the W domain did not significantly alter the ://w
The effect of PI5K on HA delivery is mediated delivery of HA indicating that expression of WA w
w
through Arp2/3- If actin comets are involved in was not generally disrupting actin-driven .jb
c
the TGN-to-apical membrane delivery of HA, then processes. As with PI5K expression, the effect of .o
rg
blockade of the N-WASP-Arp2/3 mediated actin WA on HA delivery was on rate rather than b/
y
polymerization would be expected to inhibit HA sorting, as HA surface polarity measured after g
u
e
transport. The WA domain of WASP/WAVE long chase times was not compromised (not st o
family proteins provides the binding sites for actin shown). The stimulatory and inhibitory effects of n A
p
misoolnaotimone riss aa npdo teAnrtp i2n/h3i,b iatonrd ofw Nhe-Wn AexSpPr efsusnecdt ioinn PhiIg5hKly andre pWroAdu coibnl e,H Aa ndde livwereyr e kinsettaitciss ticwaellrye ril 2, 2
0
(18,29). As a control, we expressed the W domain, significant as assessed by Student’s t-test analysis 1
9
which binds to actin monomers but not to Arp2/3 of multiple experiments (Fig. 5B). The inhibitory
(29). effect of the WA domain was specific for lipid
Infection of MDCK cells with AVs raft-enriched apical cargo, as there was no effect
encoding W or WA yielded comparable levels of of the domain on the apical delivery of p75 (Fig.
the expected protein products (W ~16 kDa; WA 5C). Moreover, expression of WA had no effect on
~18 kDa; Fig 4A). As expected, expression of WA the delivery of a basolateral marker, VSV G (Fig.
abolished PI5K-mediated formation of actin 5D).
comets, demonstrating effective inhibition of
Arp2/3 function (Table 1). We next examined the HA is associated with actin comets in MDCK
effect of the WA domain and PI5K on the cells- The data described above suggested the
morphology of polarized, filter-grown MDCK selective involvement of PI5K-stimulated, Arp2/3-
cells. Neither the WA domain nor PI5K had any dependent actin comets in the TGN-to-apical
apparent effect on cytoskeletal organization in membrane delivery of HA. To test whether newly
polarized cells (Fig 4B; similar results for W synthesized HA could be visualized in association
domain, not shown). Moreover, neither tight with actin comets, MDCK cells seeded on glass
junction morphology nor the positioning and coverslips were co-infected with AV-PI5K and
morphology of the Golgi complex was affected by AV-HA for 8 h, and then incubated at 19oC for 2.5
5
h to accumulate HA in the TGN. Samples were structure was additive, resulting in a nearly total
then warmed to 37oC for 30 min, fixed, and blockade in HA delivery to the apical membrane.
processed for indirect immunofluorescence to The dramatic inhibition of apical delivery was
visualize actin, HA, and PI5K (Fig. 6). Numerous maintained over longer chase periods (up to 4 h;
actin comets were detected in these cells, many of not shown). Treatment with NOC (either alone or
which stained positively for both PI5K and HA with WA) also inhibited delivery of HA to the
(Fig. 6, arrows). In 25 images we observed 71 basolateral surface as well (not shown). These data
comets of which 20 (28%) were positive for both suggest that HA-containing transport carriers
HA and PI5K. By contrast, in 20 images we utilize both Arp2/3 and microtubule-dependent
observed 57 comets of which only 2 (3.5%) were mechanisms to reach the apical surface, and that
positive for p75 and PI5K. Together with our ablation of both mechanisms together results in a
biochemical data, these studies provide strong profound disruption in transport.
evidence of a novel role for actin comets in
polarized biosynthetic delivery of lipid-raft DISCUSSION
associated proteins.
Here we have investigated the role of PIP
2
Efficient apical delivery of HA requires intact metabolism in polarized biosynthetic traffic.
actin and microtubule networks- The selective Expression of PI5K in polarized MDCK cells D
o
requirement for Arp2/3 in cell surface delivery of markedly stimulated TGN to apical delivery of the w
n
HA delivery suggests a role for actin dynamics in lipid-raft associated protein influenza HA whereas loa
d
apical membrane traffic. Indeed, depolymerization overexpression of Ocrl inhibited delivery. ed
of actin filaments with cytochalasin D resulted in Delivery of the non-raft associated apical protein fro
m
dramatic inhibition of HA surface delivery as p75 and of the basolateral marker VSV G were http
expected, but also had profound effects on the unaffected by PI5K. Overexpression of PI5K also ://w
polarity of HA delivery, most likely due to global caused a dramatic stimulation in actin comets that w
w
changes in actin cytoskeleton structure and cell was blocked by inhibition of Arp2/3 function upon .jb
c
morphology (data not shown). However, the actin expression of the WA domain of Scar1. .org
network in and of itself cannot confer Importantly, expression of this domain also b/
y
directionality of transport, and targeted movement selectively slowed HA transport. Moreover, gu
e
of at least some apically-destined transport carriers pharmacological stimulation of actin comet st o
in polarized cells has previously been ascribed to formation by the PKC activator PMA or inhibition n A
p
mMiocrreootuvberu,l ein-dheibpietniodne notf Armp2e/c3h afnuinscmtiso n an(3d0 a-3c4ti)n. bHyA n a-bnudt aPnIo5lK h wade rpea rvailslueal leizfefedc tisn oanss oHcAia tdioenli vwerityh. ril 2, 2
0
comet formation by the WA domain or n-butanol actin comets, confirming a role for Arp2/3- 19
treatment did not completely block HA surface mediated comet formation in HA transport.
delivery in polarized MDCK cells. We therefore Disruption of actin and microtubules together had
examined whether Arp2/3 functions a synergistic effect on apical transport, suggesting
synergistically with the microtubule network to that the two cytoskeletal assemblies normally act
enable efficient and polarized delivery of HA. in concert to direct efficient apical transport.
Polarized MDCK cells co-infected with AV-HA Together, our data suggest that apical transport of
and either control or WA-expressing AVs were raft-dependent and -independent cargo is
radiolabeled and chased at 19oC for 2 h. Prior to differentially regulated, and that raft-dependent
warming, cells were pretreated with the cargo are transported via a PKC, PIP - and
2
microtubule-depolymerizing agent nocodazole Arp2/3-dependent pathway consistent with the
(NOC) and HA delivery was assayed after a 1 h involvement of actin comets.
chase (Fig. 7). As expected, expression of WA or
treatment with NOC alone partially inhibited Role of actin in biosynthetic traffic- Numerous
delivery of HA (Fig. 7), and at this relatively short studies have documented the roles of actin in intra-
chase time, the polarity of delivery was intact (not and post-Golgi transport (35). These have ranged
shown). Interestingly, the effect of jointly from observations of the presence of actin and
disrupting Arp2/3 function and microtubule actin-associated proteins associated with the Golgi
6
complex (36-38) to more mechanistic insights into whereas carriers enriched in the non-raft apical
the potential roles of actin polymerization in protein lactase-phlorizin hydrolase traffic in
biosynthetic traffic (39). Interestingly, the ADP separate carriers in an actin-independent manner
ribosylation factor ARF1 appears to be important (3,4). Recently, the same group identified α-kinase
for actin assembly on Golgi membranes (37) and 1, which phosphorylates the motor protein myosin
this process requires coatomer-bound cdc42 and I, as a component of SI but not lactase-phlorizin
activation of the Arp2/3 complex (40,41). Cdc42 hydrolase-containing vesicles and demonstrated a
may also regulate recruitment of dynein to COPI role for this motor in apical delivery of SI (51).
vesicles (42). However, the consequences of these Whether α-kinase 1 activity is important for the
signaling cascades on intra-Golgi membrane PI5K-mediated stimulation of HA delivery we
traffic are not clear. We found no effect of observed is not known; however, in a parallel
expression of either PI5K or the WA domain of scenario, both myosin motors and N-WASP-
Scar1 on intra-Golgi transport kinetics, suggesting Arp2/3 mediated actin polymerization have been
that PIP2-and Arp2/3-mediated actin suggested as mechanisms that propel internalized
polymerization are not required for the transport or endocytic vesicles through the actin-rich cortical
maturation of cargo. cytoskeleton (52-55);(16).
ARF-dependent actin recruitment has also
been implicated in post-Golgi transport. Role of PIP2 metabolism in apical membrane D
o
Recruitment to Golgi membranes of the actin traffic- What is the site of PI5K’s role in TGN-to- w
n
binding protein cortactin was shown to be ARF- apical membrane delivery of HA? Whereas no loa
d
dependent, and disruption of this complex in BHK PI5K isoform has been localized to the Golgi ed
cells had profound effects on the surface delivery complex, the majority of this enzyme was fro
m
of VSV G without affecting intra-Golgi transport associated with actin filaments near the apical h
ttp
(43). However, our laboratory has previously surface of polarized MDCK cells close to the ://w
demonstrated that TGN export of HA occurs supranuclear Golgi complex. However, there are w
w
independently of ARF function (44). Modulation indications that some apically-destined proteins .jb
c
of actin dynamics by the clathrin- and actin- may traverse endosomal intermediates en route to .org
binding protein Hip1R has also been suggested to the cell surface (56-58), suggesting the possibility b/
y
regulate formation and release of clathrin-coated that the PI5K- and Ocrl-mediated effects on HA gu
e
vesicles from the TGN (45). transport we observed might occur at post-TGN st o
sites. Whereas the majority of Ocrl is localized to n A
p
Aroclteisn faonr da cptionl aproizleydm emriezmatbiorann ien ttrhaef ftirca-n Ssppoercti foicf tahlseo T bGeNen ( 1r5e)p,o ertneddo s(o5m9,a6l0 l)o caanldiz aLtioown eo fs yOncdrrlo hmaes ril 2, 2
0
apical proteins, and of lipid-raft associated cargo fibroblasts exhibit dramatically increased numbers 1
9
in particular, have previously been suggested. of actin comets compared with normal fibroblasts
Rozelle et al. observed that newly-synthesized HA (61,62).
was preferentially localized to the tips of short
polymers of actin reminiscent of N-WASP- Relationship between PI4P and PIP synthesis in
2
dependent comets in PI5K-overexpressing cells polarized biosynthetic traffic- Previously we
(18). In support of this, PIP2 has been suggested to found that overexpression of wild type PI4KIIIβ or
be enriched in lipid rafts (46-48) although this expression of frequenin/NCS-1, which stimulates
conclusion has recently been challenged (49,50).
PI4KIIIβ activity, inhibited apical delivery
Moreover, our observation that expression of
kinetics of HA, suggesting a negative regulatory
either PI5K or WA had no effect on apical
role for PI4P in TGN-to-apical membrane
delivery of a non-raft associated protein adds
transport (8,63). A selective role in apical
support to the idea that transport of raft-associated
membrane traffic for the PI4P-binding protein
and raft-independent proteins is differentially
Fapp2 has also been demonstrated (64). Our
regulated. Jacob et al. have previously suggested
observation here that overexpression of PI5K
that biosynthetic transport carriers containing the
stimulates apical delivery is at odds with a
lipid-raft associated hydrolase sucrase-isomaltase
simplistic precursor-product view of
(SI) traffic via actin cables to the cell surface,
7
phosphatidylinositol metabolism in which changes imaging has clearly shown that VSV G-containing
in PI4P lead to parallel changes in PIP . Indeed, transport carriers move to the plasma membrane
2
we have previously found that PI4KIIIβ-mediated on microtubule tracks (67,68). We found that
increases in cellular PI4P levels do not affect PIP disruption of actin with cytochalasin D (not
2
levels (8). It is possible that PI5K-mediated shown) or by expression of WA in concert with
conversion of PI4P to PIP might stimulate nocodazole treatment virtually abolished delivery
2
membrane traffic by relieving the inhibitory effect of HA to the apical membrane, and ultimately
of PI4P; however, this is unlikely given the high disrupted the polarity of delivery. In contrast,
levels of PI4P in the Golgi complex relative to expression of WA alone had no effect on HA
PIP . It is also possible that overexpression of polarity. Our data are reminiscent of studies by
2
PI5K causes localized changes in PI4P Maples et al., who demonstrated a concerted role
distribution; however, a more likely scenario is for actin and microtubules in basolateral-to-apical
that PI4P and PIP function independently to transcytosis of the polymeric immunoglobulin
2
modulate distinct steps in membrane transport. receptor (69). Our results do not necessarily
suggest that actin comets are obligatory for apical
Concerted cytoskeletal function in polarized delivery, but rather that they may facilitate apical
membrane traffic- Regardless of the role(s) for transport of HA under some conditions. We
actin polymerization in apical membrane traffic, it hypothesize that actin-based movement of HA- D
o
is likely that the long-range directionality of containing transport carriers facilitates their access w
n
biosynthetic membrane traffic is provided to microtubule tracks that provide the lo
a
d
ultimately by the microtubule network. Previous directionality for efficient transport to the apical ed
biochemical studies have demonstrated a role for membrane, and/or ferries transport carriers across fro
m
microtubules in polarized membrane transport in the actin rich terminal web to their site of fusion. h
ttp
MDCK cells (31,32,65,66). In addition, live cell ://w
w
w
REFERENCES .jb
c
.org
1. Schuck, S. and Simons, K. (2004) J.Cell Sci. 117, 5955-5964 b/
y
g
2. Rodriguez-Boulan, E., Kreitzer, G., and Müsch, A. (2005) Nat.Rev.Mol.Cell Biol. 6, 233- u
e
s
247 t o
n
3. Jacob, R. and Naim, H. (2001) Curr.Biol. 11, 1444-1450 Ap
4. Jacob, R., Heine, M., Alfalah, M., and Naim, H. Y. (2003) Curr.Biol. 13, 607-612 ril 2
, 2
5. Roth, M. G. (2004) Physiol Rev. 84, 699-730 0
1
9
6. Wei, Y. J., Sun, H. Q., Yamamoto, M., Wlodarski, P., Kunii, K., Martinez, M., Barylko, B.,
Albanesi, J. P., and Yin, H. L. (2002) J.Biol.Chem. 277, 46586-46593
7. Godi, A., Pertile, P., Meyers, R., Marra, P., Di Tullio, G., Iurisci, C., Luini, A., Corda, D.,
and De Matteis, M. A. (1999) Nature Cell Biol. 1, 280-287
8. Bruns, J. R., Ellis, M. A., Jeromin, A., and Weisz, O. A. (2001) J.Biol.Chem. 277, 2012-
2018
9. Weixel, K. M., Blumental-Perry, A., Watkins, S. C., Aridor, M., and Weisz, O. A. (2005)
J.Biol.Chem. 280, 10501-10508
10. Watt, S. A., Kular, G., Fleming, I. N., Downes, C. P., and Lucocq, J. M. (2002) Biochem.J.
363, 657-666
11. Jones, D. H., Morris, J. B., Morgan, C. P., Kondo, H., Irvine, R. F., and Cockcroft, S.
(2000) J.Biol.Chem. 275, 13962-13966
12. Lorra, C. and Huttner, W. B. (1999) Nature Cell Biol. 1, E113-E115
13. Huijbregts, R. P. H., Topalof, L., and Bankaitis, V. A. (2000) Traffic 1, 195-202
8
14. Dressman, M. A., Olivos-Glander, I. M., Nussbaum, R. L., and Suchy, S. F. (2000)
J.Histochem.Cytochem. 48, 179-190
15. Olivos-Glander, I. M., Janne, P. A., and Nussbaum, R. L. (1995) Am.J Hum.Genet. 57,
817-823
16. Taunton, J., Rowning, B. A., Coughlin, M. L., Wu, M., Moon, R. T., Mitchison, T. J., and
Larabell, C. A. (2000) J.Cell Biol. 148, 519-530
17. Miki, H., Miura, K., and Takenawa, T. (1996) EMBO J. 15, 5326-5335
18. Rozelle, A. L., Machesky, L. M., Yamamoto, M., Driessens, M. H. E., Insall, R. H., Roth,
M. G., Luby-Phelps, K., Marriott, G., Hall, A., and Yin, H. L. (2000) Curr.Biol. 10, 311-
320
19. Henkel, J. R., Apodaca, G., Altschuler, Y., Hardy, S., and Weisz, O. A. (1998)
Mol.Biol.Cell 8, 2477-2490
20. Apodaca, G., Katz, L. A., and Mostov, K. E. (1994) J.Cell Biol. 125, 67-86
21. Henkel, J. R., Popovich, J. L., Gibson, G. A., Watkins, S. C., and Weisz, O. A. (1999)
J.Biol.Chem. 274, 9854-9860
22. Altschuler, Y., Kinlough, C. L., Poland, P. A., Apodaca, G., Weisz, O. A., and Hughey, R.
P. (1999) Mol.Biol.Cell 11, 819-831 D
o
w
23. Yeaman, C., Burdick, D., Muesch, A., and Rodriguez-Boulan, E. (1998) Studying protein n
lo
a
sorting and transport vesicle assembly from the trans-Golgi network in intact and semi- d
e
d
intact epithelial and neuronal cells following RNA viral infection or adenovirus-mediated fro
m
gene transfer. In Celis, J. E., editor. Cell Biology: A Laboratory Handbook, 2nd edition, h
Academic Press, San Diego ttp://w
24. Lefrancois, L. and Lyles, D. S. (1982) Virology 121, 168-174 w
w
25. Yang, S. A., Carpenter, C. L., and Abrams, C. S. (2004) J.Biol.Chem. 279, 42331-42336 .jb
c
26. Orth, J. D., Krueger, E. W., Cao, H., and McNiven, M. A. (2002) .org
Proc.Natl.Acad.Sci.U.S.A. 99, 167-172 b/
y
g
27. Lee, E. and De Camilli, P. (2002) Proc.Natl.Acad.Sci.U.S.A. 99, 161-166 u
e
s
28. Chen, Y.-G., Siddhanta, A., Austin, C. D., Hammond, S. M., Sung, T.-C., Frohman, M. A., t o
n
A
Morris, A. J., and Shields, D. (1997) J.Cell Biol. 138, 495-504 p
29. Machesky, L. M. and Insall, R. H. (1998) Curr.Biol. 8, 1347-1356 ril 2
, 2
30. Noda, Y., Okada, Y., Saito, N., Setou, M., Xu, Y., Zhang, Z., and Hirokawa, N. (2001) 0
1
9
J.Cell Biol. 155, 77-88
31. Parczyk, K., Haase, W., and Kondor-Koch, C. (1989) J.Biol.Chem. 264, 16837-16846
32. Grindstaff, K. K., Bacallao, R. L., and Nelson, W. J. (1998) Mol.Biol.Cell 9, 685-699
33. Rindler, M. J., Ivanov, I. E., and Sabatini, D. D. (1987) J.Cell Biol. 104, 231-241
34. Kreitzer, G., Marmorstein, A., Okamoto, P., Vallee, R., and Rodriguez-Boulan, E. (2000)
Nature Cell Biol. 2, 125-7
35. Allan, V. J., Thompson, H. M., and McNiven, M. A. (2002) Nat.Cell Biol. 4, E236-E242
36. Heimann, K., Percival, J. M., Weinberger, R., Gunning, P., and Stow, J. L. (1999)
J.Biol.Chem. 274, 10743-10750
37. Fucini, R. V., Navarrete, A., Vadakkan, C., Lacomis, L., Erdjument-Bromage, H., Tempst,
P., and Stamnes, M. (2000) J.Biol.Chem. 275, 18824-18829
38. Matas, O. B., Martinez-Menarguez, J. A., and Egea, G. (2004) Traffic. 5, 838-846
39. Stamnes, M. (2002) Curr.Opin.Cell Biol. 14, 428-433
40. Fucini, R. V., Chen, J. L., Sharma, C., Kessels, M. M., and Stamnes, M. (2002)
Mol.Biol.Cell 13, 621-631
9
41. Dubois, T., Paleotti, O., Mironov, A. A., Fraisier, V., Stradal, T. E., De Matteis, M. A.,
Franco, M., and Chavrier, P. (2005) Nat.Cell Biol. 7, 353-364
42. Chen, J. L., Fucini, R. V., Lacomis, L., Erdjument-Bromage, H., Tempst, P., and Stamnes,
M. (2005) J.Cell Biol. 169, 383-389
43. Cao, H., Weller, S., Orth, J. D., Chen, J., Huang, B., Chen, J. L., Stamnes, M., and
McNiven, M. A. (2005) Nat.Cell Biol. 7, 483-492
44. Ellis, M. A., Miedel, M. T., Guerriero, C. J., and Weisz, O. A. (2004) J.Biol.Chem. 279,
52735-52743
45. Carreno, S., Engqvist-Goldstein, A. E., Zhang, C. X., McDonald, K. L., and Drubin, D. G.
(2004) J.Cell Biol. 165, 781-788
46. Hope, H. R. and Pike, L. J. (1996) Mol.Biol.Cell 7, 843-851
47. Laux, T., Fukami, K., Thelen, M., Golub, T., Frey, D., and Caroni, P. (2000) J.Cell Biol.
149, 1455-1471
48. Caroni, P. (2001) EMBO J. 20, 4332-4336
49. van Rheenen, J., Achame, E. M., Janssen, H., Calafat, J., and Jalink, K. (2005) EMBO J.
24, 1664-1673
50. Brough, D., Bhatti, F., and Irvine, R. F. (2005) J.Cell Sci. 118, 3019-3025 D
o
w
51. Heine, M., Cramm-Behrens, C. I., Ansari, A., Chu, H. P., Ryazanov, A. G., Naim, H. Y., n
lo
a
and Jacob, R. (2005) J.Biol.Chem. 280, 25637-25643 d
e
d
52. Morris, S. M., Arden, S. D., Roberts, R. C., Kendrick-Jones, J., Cooper, J. A., Luzio, J. P., fro
m
and Buss, F. (2002) Traffic. 3, 331-341 h
53. Benesch, S., Polo, S., Lai, F. P., Anderson, K. I., Stradal, T. E., Wehland, J., and Rottner, ttp://w
K. (2005) J.Cell Sci. 118, 3103-3115 w
w
54. Merrifield, C. J., Qualmann, B., Kessels, M. M., and Almers, W. (2004) Eur.J.Cell Biol. .jb
c
83, 13-18 .org
55. Merrifield, C. J. (2004) Trends Cell Biol. 14, 352-358 b/
y
g
56. Ang, A. L., Taguchi, T., Francis, S., Folsch, H., Murrells, L. J., Pypaert, M., Warren, G., u
e
s
and Mellman, I. (2004) J.Cell Biol. 167, 531-543 t o
n
A
57. Orzech, E., Cohen, S., Weiss, A., and Aroeti, B. (2000) J.Biol.Chem. 275, 15207-15219 p
58. Polishchuk, R., Di Pentima, A., and Lippincott-Schwartz, J. (2004) Nat.Cell Biol. 6, 297- ril 2
, 2
307 0
1
9
59. Choudhury, R., Diao, A., Zhang, F., Eisenberg, E., Saint-Pol, A., Williams, C.,
Konstantakopoulos, A., Lucocq, J., Johannes, L., Rabouille, C., Greene, L. E., and Lowe,
M. (2005) Mol.Biol.Cell 16, 3467-3479
60. Ungewickell, A., Ward, M. E., Ungewickell, E., and Majerus, P. W. (2004)
Proc.Natl.Acad.Sci.U.S.A 101, 13501-13506
61. Suchy, S. F. and Nussbaum, R. L. (2002) Am.J.Hum.Genet. 71, 1420-1427
62. Allen, P. G. (2003) Nat.Cell Biol. 5, 972-979
63. Weisz, O. A., Gibson, G. A., Leung, S.-M., Roder, J., and Jeromin, A. (2000) J.Biol.Chem.
275, 24341-24347
64. Vieira, O. V., Verkade, P., Manninen, A., and Simons, K. (2005) J.Cell Biol. 170, 521-526
65. Boll, W., Partin, J. S., Katz, A. I., Caplan, M. J., and Jamieson, J. D. (1991)
Proc.Natl.Acad.Sci.USA 88, 8592-8596
66. Breitfeld, P. P., McKinnon, W. C., and Mostov, K. E. (1990) J.Cell Biol. 111, 2365-2373
67. Hirschberg, K., Miller, C. M., Ellenberg, J., Presley, J. F., Siggia, E. D., Phair, R. D., and
Lippincott-Schwartz, J. (1998) J.Cell Biol. 143, 1485-1503
10