WAVE binds Ena/VASP for enhanced Arp2/3 complex–based actin assembly

The WAVE complex is the main activator of the Arp2/3 complex for actin filament nucleation and assembly in the lamellipodia of moving cells. Other important players in lamellipodial protrusion are Ena/VASP proteins, which enhance actin filament elongation. Here we examine the molecular coordination between the nucleating activity of the Arp2/3 complex and the elongating activity of Ena/VASP proteins for the formation of actin networks. Using an in vitro bead motility assay, we show that WAVE directly binds VASP, resulting in an increase in Arp2/3 complex–based actin assembly. We show that this interaction is important in vivo as well, for the formation of lamellipodia during the ventral enclosure event of Caenorhabditis elegans embryogenesis. Ena/VASP''s ability to bind F-actin and profilin-complexed G-actin are important for its effect, whereas Ena/VASP tetramerization is not necessary. Our data are consistent with the idea that binding of Ena/VASP to WAVE potentiates Arp2/3 complex activity and lamellipodial actin assembly.     

Dynamic relocation of the TORC1–Gtr1/2–Ego1/2/3 complex is regulated by Gtr1 and Gtr2

TORC1 regulates cellular growth, metabolism, and autophagy by integrating various signals, including nutrient availability, through the small GTPases RagA/B/C/D in mammals and Gtr1/2 in budding yeast. Rag/Gtr is anchored to the lysosomal/vacuolar membrane by the scaffold protein complex Ragulator/Ego. Here we show that Ego consists of Ego1 and Ego3, and novel subunit Ego2. The ∆ego2 mutant exhibited only partial defects both in Gtr1-dependent TORC1 activation and Gtr1 localization on the vacuole. Ego1/2/3, Gtr1/2, and Tor1/Tco89 were colocalized on the vacuole and associated puncta. When Gtr1 was in its GTP-bound form and TORC1 was active, these proteins were preferentially localized on the vacuolar membrane, whereas when Gtr1 was in its GDP-bound form, they were mostly localized on the puncta. The localization of TORC1 to puncta was further facilitated by direct binding to Gtr2, which is involved in suppression of TORC1 activity. Thus regulation of TORC1 activity through Gtr1/Gtr2 is tightly coupled to the dynamic relocation of these proteins.     

The WASH complex,an endosomal Arp2/3 activator,interacts with the Hermansky–Pudlak syndrome complex BLOC-1 and its cargo phosphatidylinositol-4-kinase type IIα

Vesicle biogenesis machinery components such as coat proteins can interact with the actin cytoskeleton for cargo sorting into multiple pathways. It is unknown, however, whether these interactions are a general requirement for the diverse endosome traffic routes. In this study, we identify actin cytoskeleton regulators as previously unrecognized interactors of complexes associated with the Hermansky–Pudlak syndrome. Two complexes mutated in the Hermansky–Pudlak syndrome, adaptor protein complex-3 and biogenesis of lysosome-related organelles complex-1 (BLOC-1), interact with and are regulated by the lipid kinase phosphatidylinositol-4-kinase type IIα (PI4KIIα). We therefore hypothesized that PI4KIIα interacts with novel regulators of these complexes. To test this hypothesis, we immunoaffinity purified PI4KIIα from isotope-labeled cell lysates to quantitatively identify interactors. Strikingly, PI4KIIα isolation preferentially coenriched proteins that regulate the actin cytoskeleton, including guanine exchange factors for Rho family GTPases such as RhoGEF1 and several subunits of the WASH complex. We biochemically confirmed several of these PI4KIIα interactions. Of importance, BLOC-1 complex, WASH complex, RhoGEF1, or PI4KIIα depletions altered the content and/or subcellular distribution of the BLOC-1–sensitive cargoes PI4KIIα, ATP7A, and VAMP7. We conclude that the Hermansky–Pudlak syndrome complex BLOC-1 and its cargo PI4KIIα interact with regulators of the actin cytoskeleton.     

α-Actinin-4/FSGS1 is required for Arp2/3-dependent actin assembly at the adherens junction

We have developed an in vitro assay to study actin assembly at cadherin-enriched cell junctions. Using this assay, we demonstrate that cadherin-enriched junctions can polymerize new actin filaments but cannot capture preexisting filaments, suggesting a mechanism involving de novo synthesis. In agreement with this hypothesis, inhibition of Arp2/3-dependent nucleation abolished actin assembly at cell-cell junctions. Reconstitution biochemistry using the in vitro actin assembly assay identified α-actinin-4/focal segmental glomerulosclerosis 1 (FSGS1) as an essential factor. α-Actinin-4 specifically localized to sites of actin incorporation on purified membranes and at apical junctions in Madin-Darby canine kidney cells. Knockdown of α-actinin-4 decreased total junctional actin and inhibited actin assembly at the apical junction. Furthermore, a point mutation of α-actinin-4 (K255E) associated with FSGS failed to support actin assembly and acted as a dominant negative to disrupt actin dynamics at junctional complexes. These findings demonstrate that α-actinin-4 plays an important role in coupling actin nucleation to assembly at cadherin-based cell-cell adhesive contacts.     

IKK1/2 protect human cells from TNF-mediated RIPK1-dependent apoptosis in an NF-κB-independent manner

TNF signaling is directly linked to cancer development and progression. A broad range of tumor cells is able to evade cell death induced by TNF impairing the potential anti-cancer value of TNF in therapy. Although sensitizing cells to TNF-induced death therefore has great clinical implications, detailed mechanistic insights into TNF-mediated human cell death still remain unknown. Here, we analyzed human cells by applying CRISPR/Cas9n to generate cells deficient of IKK1, IKK2, IKK1/2 and RELA. Despite stimulation with TNF resulted in impaired NF-κB activation in all genotypes compared to wildtype cells, increased cell death was observable only in IKK1/2-double-deficient cells. Cell death could be detected by Caspase-3 activation and binding of Annexin V. TNF-induced programmed cell death in IKK1/2^?/? cells was further shown to be mediated via RIPK1 in a predominantly apoptotic manner. Our findings demonstrate the IKK complex to protect from TNF-induced cell death in human cells independently to NF-κB RelA suggesting IKK1/2 to be highly promising targets for cancer therapy.     

Collagen/β1 integrin signaling up-regulates the ABCC1/MRP-1 transporter in an ERK/MAPK-dependent manner

The mechanisms by which β1 integrins regulate chemoresistance of cancer cells are still poorly understood. In this study, we report that collagen/β1 integrin signaling inhibits doxorubicin-induced apoptosis of Jurkat and HSB2 leukemic T-cells by up-regulating the expression and function of the ATP-binding cassette C 1 (ABCC1) transporter, also known as multidrug resistance-associated protein 1. We find that collagen but not fibronectin reduces intracellular doxorubicin content and up-regulates the expression levels of ABCC1. Inhibition and knockdown studies show that up-regulation of ABCC1 is necessary for collagen-mediated reduction of intracellular doxorubicin content and collagen-mediated inhibition of doxorubicin-induced apoptosis. We also demonstrate that activation of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase signaling pathway is involved in collagen-induced reduction of intracellular doxorubicin accumulation, collagen-induced up-regulation of ABCC1 expression levels, and collagen-mediated cell survival. Finally, collagen-mediated up-regulation of ABCC1 expression and function also requires actin polymerization. Taken together, our results indicate for the first time that collagen/β1 integrin/ERK signaling up-regulates the expression and function of ABCC1 and suggest that its activation could represent an important pathway in cancer chemoresistance. Thus simultaneous targeting of collagen/β1 integrin and ABCC1 may be more efficient in preventing drug resistance than targeting each pathway alone.     

Arabidopsis GCP3-interacting protein 1/MOZART 1 is an integral component of the γ-tubulin-containing microtubule nucleating complex

Microtubules in eukaryotic cells are nucleated from ring-shaped complexes that contain γ-tubulin and a family of homologous γ-tubulin complex proteins (GCPs), but the subunit composition of the complexes can vary among fungi, animals and plants. Arabidopsis GCP3-interacting protein 1 (GIP1), a small protein with no homology to the GCP family, interacts with GCP3 in vitro, and is a plant homolog of vertebrate mitotic-spindle organizing protein associated with a ring of γ-tubulin 1 (MOZART1), a recently identified component of the γ-tubulin complex in human cell lines. In this study, we characterized two closely related Arabidopsis GIP1s: GIP1a and GIP1b. Single mutants of gip1a and gip1b were indistinguishable from wild-type plants, but their double mutant was embryonic lethal, and showed impaired development of male gametophytes. Functional fusions of GIP1a with green fluorescent protein (GFP) were used to purify GIP1a-containing complexes from Arabidopsis plants, which contained all the subunits (except NEDD1) previously identified in the Arabidopsis γ-tubulin complexes. GIP1a and GIP1b interacted specifically with Arabidopsis GCP3 in yeast. GFP-GIP1a labeled mitotic microtubule arrays in a pattern largely consistent with, but partly distinct from, the localization of the γ-tubulin complex containing GCP2 or GCP3 in planta. In interphase cortical arrays, the labeled complexes were preferentially recruited to existing microtubules, from which new microtubules were efficiently nucleated. However, in contrast to complexes labeled with tagged GCP2 or GCP3, their recruitment to cortical areas with no microtubules was rarely observed. These results indicate that GIP1/MOZART1 is an integral component of a subset of the Arabidopsis γ-tubulin complexes.     

PCAF and SIRT1 modulate βTrCP1 protein stability in an acetylation-dependent manner

正Ubiquitination plays critical roles in regulating various physiological events,such as protein degradation,activation,secretion,sorting and trafficking (Dang et al.,2021).The ubiquitination process involves three major steps,catalyzed by ubiquitin-activating enzymes (E1s),ubiquitin-conjugating enzymes (E2s),and ubiquitin ligases (E3s),respectively (Scheffner et al.,1995).     

GSK3β is involved in the relief of mitochondria pausing in a Tau-dependent manner

Mitochondrial trafficking deficits have been implicated in the pathogenesis of several neurological diseases, including Alzheimer's disease (AD). The Ser/Thre kinase GSK3β is believed to play a fundamental role in AD pathogenesis. Given that GSK3β substrates include Tau protein, here we studied the impact of GSK3β on mitochondrial trafficking and its dependence on Tau protein. Overexpression of GSK3β in neurons resulted in an increase in motile mitochondria, whereas a decrease in the activity of this kinase produced an increase in mitochondria pausing. These effects were dependent on Tau proteins, as Tau (-/-) neurons did not respond to distinct GSK3β levels. Furthermore, differences in GSK3β expression did not affect other parameters like mitochondria velocity or mitochondria run length. We conclude that GSK3B activity regulates mitochondrial axonal trafficking largely in a Tau-dependent manner.     

IRES-mediated translation of cellular messenger RNA operates in eIF2α- independent manner during stress

Physiological and pathophysiological stress attenuates global translation via phosphorylation of eIF2α. This in turn leads to the reprogramming of gene expression that is required for adaptive stress response. One class of cellular messenger RNAs whose translation was reported to be insensitive to eIF2α phosphorylation-mediated repression of translation is that harboring an Internal Ribosome Entry Site (IRES). IRES-mediated translation of several apoptosis-regulating genes increases in response to hypoxia, serum deprivation or gamma irradiation and promotes tumor cell survival and chemoresistance. However, the molecular mechanism that allows IRES-mediated translation to continue in an eIF2α-independent manner is not known. Here we have used the X-chromosome linked Inhibitor of Apoptosis, XIAP, IRES to address this question. Using toeprinting assay, western blot analysis and polysomal profiling we show that the XIAP IRES supports cap-independent translation when eIF2α is phosphorylated both in vitro and in vivo. During normal growth condition eIF2α-dependent translation on the IRES is preferred. However, IRES-mediated translation switches to eIF5B-dependent mode when eIF2α is phosphorylated as a consequence of cellular stress.     

A WAVE2–Arp2/3 actin nucleator apparatus supports junctional tension at the epithelial zonula adherens

The epithelial zonula adherens (ZA) is a specialized adhesive junction where actin dynamics and myosin-driven contractility coincide. The junctional cytoskeleton is enriched in myosin II, which generates contractile force to support junctional tension. It is also enriched in dynamic actin filaments, which are replenished by ongoing actin assembly. In this study we sought to pursue the relationship between actin assembly and junctional contractility. We demonstrate that WAVE2–Arp2/3 is a major nucleator of actin assembly at the ZA and likely acts in response to junctional Rac signaling. Furthermore, WAVE2–Arp2/3 is necessary for junctional integrity and contractile tension at the ZA. Maneuvers that disrupt the function of either WAVE2 or Arp2/3 reduced junctional tension and compromised the ability of cells to buffer side-to-side forces acting on the ZA. WAVE2–Arp2/3 disruption depleted junctions of both myosin IIA and IIB, suggesting that dynamic actin assembly may support junctional tension by facilitating the local recruitment of myosin.     

Concerted removal of the Erb1–Ytm1 complex in ribosome biogenesis relies on an elaborate interface

The complicated process of eukaryotic ribosome biogenesis involves about 200 assembly factors that transiently associate with the nascent pre-ribosome in a spatiotemporally ordered way. During the early steps of 60S subunit formation, several proteins, collectively called A₃ cluster factors, participate in the removal of the internal transcribed spacer 1 (ITS1) from 27SA₃ pre-rRNA. Among these factors is the conserved hetero-trimeric Nop7–Erb1–Ytm1 complex (or human Pes1–Bop1–Wdr12), which is removed from the evolving pre-60S particle by the AAA ATPase Rea1 to allow progression in the pathway. Here, we clarify how Ytm1 and Erb1 interact, which has implications for the release mechanism of both factors from the pre-ribosome. Biochemical studies show that Ytm1 and Erb1 bind each other via their ß-propeller domains. The crystal structure of the Erb1–Ytm1 heterodimer determined at 2.67Å resolution reveals an extended interaction surface between the propellers in a rarely observed binding mode. Structure-based mutations in the interface that impair the Erb1–Ytm1 interaction do not support growth, with specific defects in 60S subunit synthesis. Under these mutant conditions, it becomes clear that an intact Erb1–Ytm1 complex is required for 60S maturation and that loss of this stable interaction prevents ribosome production.     

Ethylene-induced stomatal closure is mediated via MKK1/3–MPK3/6 cascade to EIN2 and EIN3

Mitogen-activated protein kinases (MPKs) play essential roles in guard cell signaling, but whether MPK cascades participate in guard cell ethylene signaling and interact with hydrogen peroxide (H₂O₂), nitric oxide (NO), and ethylene-signaling components remain unclear. Here, we report that ethylene activated MPK3 and MPK6 in the leaves of wild-type Arabidopsis thaliana as well as ethylene insensitive2 (ein2), ein3, nitrate reductase1 (nia1), and nia2 mutants, but this effect was impaired in ethylene response1 (etr1), nicotinamide adenine dinucleotide phosphate oxidase AtrbohF, mpk kinase1 (mkk1), and mkk3 mutants. By contrast, the constitutive triple response1 (ctr1) mutant had constitutively active MPK3 and MPK6. Yeast two-hybrid, bimolecular fluorescence complementation, and pull-down assays indicated that MPK3 and MPK6 physically interacted with MKK1, MKK3, and the C-terminal region of EIN2 (EIN2 CEND). mkk1, mkk3, mpk3, and mpk6 mutants had typical levels of ethylene-induced H₂O₂ generation but impaired ethylene-induced EIN2 CEND cleavage and nuclear translocation, EIN3 protein accumulation, NO production in guard cells, and stomatal closure. These results show that the MKK1/3–MPK3/6 cascade mediates ethylene-induced stomatal closure by functioning downstream of ETR1, CTR1, and H₂O₂ to interact with EIN2, thereby promoting EIN3 accumulation and EIN3-dependent NO production in guard cells.     

Plasmodium falciparum RuvB1 is an active DNA helicase and translocates in the 5′–3′ direction

RuvB family of protein contains two similar kinds of proteins i.e. RuvB1 and RuvB2 from yeast to human. These proteins belong to the AAA + class of proteins and are critical components of several multiprotein complexes involved in diverse cellular activities. There are two RuvB proteins annotated in the Plasmodium database but the identification of the third protein recently by our lab has raised the question why Plasmodium falciparum contains three RuvB proteins instead of two. Hence the biochemical characterizations of these proteins have become essential to understand the role of these proteins in the malaria parasite. Recently we have reported the characterization of the recombinant PfRuvB3, which contains ATPase activity but lacks DNA helicase activity. In the present study we report the phylogenetic analysis and detailed biochemical characterization of one of the other RuvB homologue RuvB1 from P. falciparum. PfRuvB1 shows considerable homology with human as well as yeast RuvB1 and contains Walker motif A and Walker motif B. The activity analysis of this protein revealed that PfRuvB1 is an ATPase and this activity increased significantly in the presence of ss-DNA. PfRuvB1 also contains DNA helicase activity and translocates preferentially in 5′ to 3′ direction. In vivo investigation of PfRuvB1 revealed that it is constitutively expressed during all the stages of intraerythrocytic cycle of P. falciparum and localizes mainly to the nucleus. These studies will make important contribution in understanding the role of RuvB protein in P. falciparum.     

C16-Ceramide-induced F-actin regulation stimulates mouse embryonic stem cell migration: Involvement of N-WASP/Cdc42/Arp2/3 complex and cofilin-1/α-actinin

Ceramide, a major structural element in the cellular membrane, is a key regulatory factor in various cellular behaviors that are dependent on ceramide-induced association of specific proteins. However, molecular mechanisms that regulate ceramide-induced embryonic stem cell (ESC) migration are still not well understood. Thus, we investigated the effect of ceramide on migration and its related signal pathways in mouse ESCs. Among ceramide species with different fatty acid chain lengths, C₁₆-Cer increased migration of mouse ESCs in a dose- (≥ 1 μM) and time-dependent (≥ 8 h) manners, as determined by the cell migration assay. C₁₆-Cer (10 μM) increased protein-kinase C (PKC) phosphorylation. Subsequently, C₁₆-Cer increased focal adhesion kinase (FAK) and Paxillin phosphorylation, which were inhibited by PKC inhibitor Bisindolylmaleimide I (1 μM). When we examined for the downstream signaling molecules, C₁₆-Cer activated small G protein (Cdc42) and increased the formation of complex with Neural Wiskott-Aldrich Syndrome Protein (N-WASP)/Cdc42/Actin-Related Protein 2/3 (Arp2/3). This complex formation was disrupted by FAK- and Paxillin-specific siRNAs. Furthermore, C₁₆-Cer-induced increase of filamentous actin (F-actin) expression was inhibited by Cdc42-, N-WASP-, and Arp2/3-specific siRNAs, respectively. Indeed, C₁₆-Cer increased cofilin-1/F-actin interaction or F-actin/α-actinin-1 and α-actinin-4 interactions in the cytoskeleton compartment, which was reversed by Cdc42-specific siRNA. Finally, C₁₆-Cer-induced increase of cell migration was inhibited by knocking down each signal pathway-related molecules with siRNA or inhibitors. In conclusion, C₁₆-Cer enhances mouse ESC migration through the regulation of PKC and FAK/Paxillin-dependent N-WASP/Cdc42/Arp2/3 complex formation as well as through promoting the interaction between cofilin-1 or α-actinin-1/-4 and F-actin.     

ARG1 and ARL2 form an actin-based gravity-signaling chaperone complex in root statocytes?

Plants are acutely sensitive to the directional information provided by gravity. They have evolved statocytes, which are specialized cells that sense gravity and, upon integration of the corresponding information with that of other environmental stimuli, control the growth behavior of their organs. The cellular mechanisms that allow statocytes to sense and transduce gravitational information likely involve detecting the sedimentation of, or the tension/pressure exerted by, starch-filled amyloplasts—the presumptive statoliths—within their cytoplasm. Gravity signaling in root statocytes controls the direction of transport of signaling compounds, especially auxin, across the root cap, establishing a lateral gradient that is transmitted to cells in the elongation zone and results in gravitropic curvature. The Arabidopsis J-domain proteins ARG1 and ARL2 function as gravity-signal transducers in root statocytes. In the January issue of The Plant Journal, we reported that ARG1 and ARL2 function non-redundantly in a common gravity signaling pathway required for accumulation of the auxin efflux facilitator PIN3 on the new bottom side of statocytes following gravity stimulation, and lateral redistribution of auxin toward the new lower flank of stimulated roots. Here we present data suggesting that ARG1 physically associates with ARL2, the J-domain co-chaperone HSC70, and actin in vivo. We briefly discuss potential mechanisms by which ARG1 and ARL2 might function in gravity signaling in light of this information.Key words: gravitropism, statocytes, actin, HSC70/HSP70, auxinGravitropic growth in plants requires the ability to sense an organ''s orientation within the gravity field, transduce that information into a biochemical signal, and mount a differential cellular-elongation response that corrects growth to follow a defined gravity set point angle. Gravity sensation in roots occurs mainly in statocytes of the root cap columella, either via detection of the position or movement of, or the pressure exerted by, amyloplasts within these cells, or by perceiving the pressure exerted by the statocyte''s protoplast within its cell wall.^1,2 Several models address the mechanism by which statocytes use amyloplasts as gravity susceptors. One model postulates that gravity signal transduction initiates when sedimenting amyloplasts promote the opening of mechano-sensitive ion channels, either directly or through interaction with the actin cytoskeleton. Alternatively, signal transduction may initiate upon sedimentation, when amyloplast-borne ligands interact with receptors located on sensitive structures within the statocytes.¹ Nevertheless, the activated pathway promotes a fast and transient cytoplasmic alkalinization and redistribution of the auxin efflux carrier PIN3 to the lower membrane of the statocytes. These changes contribute to lateral, downward, redistribution of auxin across the cap, resulting in the formation of a lateral auxin gradient that, upon transmission to the elongation zone, promotes tip curvature (reviewed in ref. ¹).Along with experiments demonstrating the importance of amyloplasts in gravity susception, genetic analysis in Arabidopsis has identified ARG1 and ARL2 as gravity signal transducers in root and hypocotyl statocytes.^3–6 ARG1 and ARL2 function in a gravity-signaling pathway that links gravistimulation to the accumulation of PIN3 within the PM at the lower side of the statocytes and a redistribution of auxin across the cap.^4–6 ARG1 and ARL2 are membrane-associated proteins. While ARG1 localizes throughout the endosomal/secretory pathway, ARL2 associates primarily with the PM, and both proteins are found at the cell plate during cytokinesis.^4,6 ARG1 and ARL2 contain J-domains near their N-termini. J-domain proteins typically function as molecular co-chaperones by interacting with HSC70, coupling substrate binding to HSC70 ATPase activity.¹⁰ A direct role for HSC70 in gravity signaling has not been reported, though proteomics approaches have identified cytosolic HSC70 isoforms as gravity-regulated proteins within Arabidopsis root tips.^7,8The C-termini of ARG1 and ARL2 contain putative coiled-coil domains with similarity to predicted coiled-coils found in proteins that interact with the cytoskeleton, including Rho-associated kinases, myosin, heavy chain kinesins and tropomyosins.³ Interestingly, a biochemical fractionation experiment detected possible interaction between ARG1 and polymerized actin in plant extracts.⁴ Furthermore, proteomic analyses of root gravitropism also identified actin as a gravity-regulated protein^7,8 whereas other studies strongly suggest a role for the actin cytoskeleton in the modulation of gravity signaling in roots.⁹To investigate possible in vivo interactions between ARG1, ARL2, HSC70 and/or actin, we generated transgenic plants expressing GFP fusions to ARG1 or ARL2, in the arg1-2 or arl2-3 mutant backgrounds (Ws alleles), and used them in co-immunoprecipitation experiments. All constructs were tested for functional rescue prior to use. Functional GFP fusions to ARG1 or ARL2 were isolated from plant extracts using immobilized anti-GFP antibodies. Initial experiments indicated that both N and C-terminal GFP fusions to ARG1 and C-terminal fusions to ARL2 could be purified by this method (Fig. 1A). However, we consistently noticed lower relative abundance of ARL2-GFP purified from plants compared to ARG1-GFP, even when both were expressed under the same promoter (Fig. 1A). This is consistent with lower fluorescence intensity and fusion protein abundance of ARL2-GFP compared to ARG1-GFP in the many transgenic lines analyzed (data not shown).Open in a separate windowFigure 1HSC70, actin and ARL2 co-purify with ARG1 from plant extracts. Total protein extracts from four-week-old liquid grown plants of the indicated genotypes (top) were affinity-purified using anti-GFP antibodies (gift of Richard Vierstra, UW-Madison) cross-linked to Protein A-Sepharose beads and detected with the indicated antibodies. In (A) the affinity-purified eluate was probed with anti-GFP antibody to indicate the presence of both full-length GFP fusions and breakdown products. In (B) western blots of total protein extracts (Input, In) and affinity-purified eluates (E) were probed with anti-HSC70 (diluted 1:10,000; Stressgen, San Diego, CA), monoclonal anti-actin (diluted 1:1000; clone C4 from Chemicon Int., Temecula, CA), or anti-a-tubulin antibodies (Sigma, St Louis, MO). In (C) western blots of affinity-purified eluates (top) were probed with two anti-ARG1 antibodies raised in rabbits against a bacterially expressed His6-ARG1 protein fusion (anti-ARG1 I) and against a peptide corresponding to the 15 C-terminal residues of ARG1 (anti-ARG1 II), both used at 1:1000 dilutions, as well as with a polyclonal anti-ARL2 antibody raised in rabbits against a synthetic ARL2 peptide, used at 1:3000 dilution. Note that anti-ARG1 II did not recognize the ARG1-GFP protein probably because the corresponding C-terminal epitope was masked in this C-terminal fusion. A western blot of total protein extracts used for affinity purification (In, bottom) was probed with anti-HSP70 antibody (gift of Elizabeth Craig, UW-Madison; 1:20,000 dilution) to show relative amounts of protein prior to extraction. Experimental procedures. Plants expressing ARG1::GFP-ARG1 and 35S::ARL2-GFP are described elsewhere (reviewed in refs. ⁴ and ⁶, respectively). Plants expressing 35S::ARG1-GFP were generated by transformation with a T-DNA derived from the binary vector pK7FWG2²⁵ containing the ARG1 cDNA, whereas plants expressing AtMDR1::GFP were a gift from Edgar Spalding (UW-Madison). For immunoprecipitation, anti-GFP serum was cross-linked to Protein A Sepharose (Amersham, Piscataway, NJ) using the cross-linking reagent disuccinimidyl suberate according to the manufacturer''s recommendations (Pierce, Rockford, Ill.). Extracts from liquid-grown plants were generated by grinding in 1:1 (vol:wt) ice cold grinding buffer [20 mM Tris, 1 mM EDTA, 100 mM NaCl, 0.1% Triton X-100 pH7.5, containing 1:100 dilution of protease inhibitor cocktail (Sigma, or Calbiochem, San Diego, CA) and 1 mM phenylmethylsulphonyl fluoride] in a mortar, then adding more buffer to a ratio of 4:1 (vol:wt). Homogenates were cleared by filtering through Miracloth (Calbiochem) and spinning twice at 13,000 g for 15 min each to pellet cellular debris. Protein concentration in the resulting supernatant was then quantified by a modified Lowry assay, using BSA as a standard. Extracts were gently rocked with anti-GFP beads for one hour at room temperature. The beads were washed extensively (five times with ≥5× bead vol.) in extraction buffer, and the bound protein was eluted with low pH (0.1 M glycine pH 2.5) into a small amount of neutralizing 1 M tris buffer (pH 8.0), precipitated with TCA, separated by SDS-PAGE, blotted to PVDF membrane (Millipore, Billerica, MA), and probed with the appropriate antibodies. Positive signals were detected using HRP-conjugated secondary antibodies at 1:20,000 (Sigma).Proteins that interact with ARG1 or ARL2 are expected to bind to the anti-GFP-coated beads in this assay, along with their GFP-ARG1 or GFP-ARL2 partner, and to co-elute with them upon washing the beads with a low-pH solution. Their identity can be revealed by western blot analysis of bead eluates, using specific antibodies. Figure 1B shows that HSC70 co-purifies with ARG1-GFP and GFP-ARG1 when extracts from plants expressing these fusions are tested in this assay. However, HSC70 was not present in the eluate when plants expressing GFP alone under the control of the CaMV35S or AtMDR1 ectopic promoters were tested (Fig. 1B). Interestingly, we did not detect HSC70 in bead eluates when ARL2-GFP-expressing plants were subjected to this co-immunoprecipitation assay, suggesting that ARG1 and ARL2 differ in their affinity for HSC70. It should however be noted that the low amount of ARL2-GFP precipitated in these experiments does not allow for direct comparison (Fig. 1B). These results indicate an in vivo interaction between ARG1 and HSC70, suggesting that ARG1 is a bona fide co-chaperone.Figure 1B also shows that actin is present specifically in immunocomplexes containing ARG1. In a parallel experiment, ARL2-GFP precipitates did not contain detectable actin (Fig. 1B). However, here again, the low amount of ARL2 in these precipitates does not allow direct comparison to ARG1. Together these data suggest that ARG1 physically associates with actin in vivo, even though they do not address whether this association involves monomeric or polymerized actin. Interestingly, the biochemical fractionation experiments reported by Boonsirichai et al. (2003) suggested that some ARG1 might associate with polymerized actin in vivo.Plants with mutations in ARG1, ARL2, or both, result in very similar gravity signaling defects, demonstrating that ARG1 and ARL2 function as non-redundant members of the same signaling pathway.⁵ One interpretation of this data is that ARG1 and ARL2 function in the same signaling complex, the presence of both being required for complex activity in gravity signaling. In support of this model, we have detected an in vivo interaction between ARG1 and ARL2 by immunoprecipitation. Figure 1C shows that endogenous ARG1 (∼45 kDa) co-immunoprecipitates with ARL2-GFP from extracts derived from plants expressing 35S::ARL2-GFP, but not from extracts derived from negative control plants. We were not surprised by the failure to detect ARG1 in precipitates from plants expressing ARL2-GFP under the control of its endogenous promoter (ARL2::ARL2-GFP). Indeed, its expression is restricted to a few cells—therefore, the protein is present at low abundance in whole-plant extracts.⁶ Similarly, native ARL2 was not detected in ARG1 immunoprecipitates likely due to the low level of endogenous ARL2 expression. Together, these results suggest that ARG1 and ARL2 associate in a complex in vivo, consistent with the function of these proteins in a common signaling pathway.⁵The sub-cellular localization of a putative ARG1- and ARL2-containing complex is unknown, but is likely associated with the PM of root statocytes, where ARL2-GFP localizes in cells expressing ARL2::ARL2-GFP.⁶ Such a complex may serve as a membrane-associated signaling module that recruits cytosolic HSC70 to a membrane site in a gravity-regulated manner. In agreement with this model, we found that HSP70 increases in abundance in a biochemical fraction that includes membrane-associated proteins extracted from Arabidopsis root tips within 10 min of a gravistimulus.⁸ A similar recruitment model has been proposed for J-protein function in several HSC70-mediated processes, including the regulation of vesicle dynamics and signaling by HSC70.¹⁰ In animal systems, HSC70 activity is required for several steps in clatherin-dependent vesicle recycling,¹¹ a process that appears to mediate PIN protein trafficking¹². Recruitment of HSC70 activity to clatherin-coated vesicles is mediated mainly by the J-domain protein auxillin, which has paralogs other than ARG1 and ARL2 in plants.¹⁰ HSC70 function is also required for Ca²⁺-induced exocytosis in Drosophila, and depends on the J-domain-containing cysteine-string protein, apparent homologs of which are absent from plants.¹³ GRV2, a locus required for normal shoot tropisms, encodes a membrane-associated J-domain protein homologous to animal RME-8.¹⁴ RME-8 is a membrane-associated J-domain protein that interacts with cytosolic HSC70 and is required for endocytosis and endosomal trafficking in D. melanogaster and C. elegans.^15,16 GRV2 appears to regulate dynamics of late vacuolar endosomes,¹⁷ which is important for gravity signaling in shoots but not roots.¹⁸ GRV2/RME-8, auxillin, and cysteine-string proteins are each specific for endosomal trafficking involving particular molecules or vesicles, thus illustrating the specificity that J-domain proteins can confer to HSC70-mediated processes.One possibility is that the putative ARG1/ARL2 complex recruits HSC70 to alter actin dynamics in response to gravistimulation. The actin network in root statocytes appears to be intimately associated with the mechanism of gravity sensation, as treatments with low concentrations of the actin-depolymerizing drug Latrunculin B enhances gravity signaling events including amyloplast sedimentation, alkalinization of the statocyte cytoplasm, with subsequent enhancement of both auxin redistribution and organ curvature.⁹ arg1-2 mutants are deficient in gravity-induced statocyte alkalinization and auxin redistribution, suggesting that ARG1 and microfilaments regulate this gravity signaling response.⁴The lateral polarization of the statocytes upon gravistimulation likely involves dramatic changes in vesicle trafficking. Mechanisms that lead to accumulation of PIN3 upon the new bottom side of root statocytes are unknown, but require ARG1 and ARL2 either directly or as upstream signaling components. The prominent localization of ARG1 and ARL2 to the cell plate during cytokinesis suggests that they are present in areas of intense vesicle dynamics. The recent discovery that members of an Arabidopsis retromer complex are required for cell polarization, PIN trafficking, and gravitropic growth suggests they may function in redirection of auxin following gravistimulation.^19,20 Animal homologs of these retromer components have been identified in association with clathrin-coated vesicles,²¹ and disruption of Drosophila Vps35 or human SNX9 affect endocytosis and actin organization.^22,23 The mechanisms of clathrin-dependent endocytosis are unresolved, but appear to depend on dynamic cortical microfilaments in animals and yeast suggesting that similar mechanisms may function in plants.²⁴ ARG1 and ARL2 may mediate statocyte polarization via regulating actin and/or vesicle dynamics. Of course, we have not yet excluded the possibility that ARG1 and ARL2 might, in fact, transduce the gravity signal in an HSC70 and actin-independent manner through interactions with yet unidentified molecules. In this case, the interactions identified in this study would be relevant to other, yet uncharacterized, functions potentially associated with ARG1 and ARL2 in plants.^4,5 Work aimed at identifying additional ARG1 and ARL2-interacting proteins is underway.