scholarly journals In vitro reconstitution reveals phosphoinositides as cargo-release factors and activators of the ARF6 GAP ADAP1

2020 ◽  
Vol 118 (1) ◽  
pp. e2010054118
Author(s):  
Christian Duellberg ◽  
Albert Auer ◽  
Nikola Canigova ◽  
Katrin Loibl ◽  
Martin Loose
Keyword(s):  
Molecular Motors ◽  
Small Gtpase ◽  
Complex Dynamic ◽  
Gtp Hydrolysis ◽  
Dendrite Branching ◽  
Precise Control ◽  
In Vitro Reconstitution ◽  
Adp Ribosylation Factor ◽  
Dynamic Interplay

The differentiation of cells depends on a precise control of their internal organization, which is the result of a complex dynamic interplay between the cytoskeleton, molecular motors, signaling molecules, and membranes. For example, in the developing neuron, the protein ADAP1 (ADP-ribosylation factor GTPase-activating protein [ArfGAP] with dual pleckstrin homology [PH] domains 1) has been suggested to control dendrite branching by regulating the small GTPase ARF6. Together with the motor protein KIF13B, ADAP1 is also thought to mediate delivery of the second messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP3) to the axon tip, thus contributing to PIP3polarity. However, what defines the function of ADAP1 and how its different roles are coordinated are still not clear. Here, we studied ADAP1’s functions using in vitro reconstitutions. We found that KIF13B transports ADAP1 along microtubules, but that PIP3as well as PI(3,4)P2act as stop signals for this transport instead of being transported. We also demonstrate that these phosphoinositides activate ADAP1’s enzymatic activity to catalyze GTP hydrolysis by ARF6. Together, our results support a model for the cellular function of ADAP1, where KIF13B transports ADAP1 until it encounters high PIP3/PI(3,4)P2concentrations in the plasma membrane. Here, ADAP1 disassociates from the motor to inactivate ARF6, promoting dendrite branching.

scholarly journals Bacterial Microtubules Exhibit Polarized Growth, Mixed-Polarity Bundling, and Destabilization by GTP Hydrolysis

2017 ◽  
Author(s):  
César Díaz-Celis ◽  
Viviana I. Risca ◽  
Felipe Hurtado ◽  
Jessica K. Polka ◽  
Scott D. Hansen ◽  
...  
Keyword(s):  
Molecular Motors ◽  
Intracellular Transport ◽  
Complex Dynamics ◽  
Critical Concentration ◽  
Polarized Growth ◽  
Gtp Hydrolysis ◽  
Filament Dynamics ◽  
Mixed Polarity ◽  
Self Association

AbstractBacteria of the genusProsthecobacterexpress homologs of eukaryotic α-and β-tubulin, called BtubA and BtubB, that have been observed to assemble into bacterial microtubules (bMTs). ThebtubABgenes likely entered theProsthecobacterlineage via horizontal gene transfer and may derive from an early ancestor of the modern eukaryotic microtubule (MT). Previous biochemical studies revealed that BtubA/B polymerization is GTP-dependent and reversible and that BtubA/B folding does not require chaperones. To better understand bMT behavior and gain insight into the evolution of microtubule dynamics, we characterizedin vitrobMT assembly using a combination of polymerization kinetics assays, and microscopy. Like eukaryotic microtubules, bMTs exhibit polarized growth with different assembly rates at each end. GTP hydrolysis stimulated by bMT polymerization drives a stochastic mechanism of bMT disassembly that occurs via polymer breakage. We also observed treadmilling (continuous addition and loss of subunits at opposite ends) of bMT fragments. Unlike MTs, polymerization of bMTs requires KCl, which reduces the critical concentration for BtubA/B assembly and induces bMTs to form stable mixed-orientation bundles in the absence of any additional bMT-binding proteins. Our results suggest that at potassium concentrations resembling that inside the cytoplasm ofProsthecobacter, bMT stabilization through self-association may be a default behavior. The complex dynamics we observe in both stabilized and unstabilized bMTs may reflect common properties of an ancestral eukaryotic tubulin polymer.ImportanceMicrotubules are polymers within all eukaryotic cells that perform critical functions: they segregate chromosomes in cell division, organize intracellular transport by serving as tracks for molecular motors, and support the flagella that allow sperm to swim. These functions rely on microtubules remarkable range of tunable dynamic behaviors. Recently discovered bacterial microtubules composed of an evolutionarily related protein are evolved from a missing link in microtubule evolution, the ancestral eukaryotic tubulin polymer. Using microscopy and biochemical approaches to characterize bacterial microtubules, we observed that they exhibit complex and structurally polarized dynamic behavior like eukaryotic microtubules, but differ in how they self-associate into bundles and become destabilized. Our results demonstrate the diversity of mechanisms that microtubule-like filaments employ to promote filament dynamics and monomer turnover.

scholarly journals Discrete Determinants in ArfGAP2/3 Conferring Golgi Localization and Regulation by the COPI Coat

2009 ◽  
Vol 20 (3) ◽  
pp. 859-869 ◽  
Author(s):  
Lena Kliouchnikov ◽  
Joëlle Bigay ◽  
Bruno Mesmin ◽  
Anna Parnis ◽  
Moran Rawet ◽  
...  
Keyword(s):  
Lipid Membranes ◽  
Membrane Curvature ◽  
In Vitro Assays ◽  
Minor Role ◽  
Gtp Hydrolysis ◽  
Gtpase Activating Proteins ◽  
Golgi Localization ◽  
Adp Ribosylation Factor ◽  
Fusion Approach

From yeast to mammals, two types of GTPase-activating proteins, ArfGAP1 and ArfGAP2/3, control guanosine triphosphate (GTP) hydrolysis on the small G protein ADP-ribosylation factor (Arf) 1 at the Golgi apparatus. Although functionally interchangeable, they display little similarity outside the catalytic GTPase-activating protein (GAP) domain, suggesting differential regulation. ArfGAP1 is controlled by membrane curvature through its amphipathic lipid packing sensor motifs, whereas Golgi targeting of ArfGAP2 depends on coatomer, the building block of the COPI coat. Using a reporter fusion approach and in vitro assays, we identified several functional elements in ArfGAP2/3. We show that the Golgi localization of ArfGAP3 depends on both a central basic stretch and a carboxy-amphipathic motif. The basic stretch interacts directly with coatomer, which we found essential for the catalytic activity of ArfGAP3 on Arf1-GTP, whereas the carboxy-amphipathic motif interacts directly with lipid membranes but has minor role in the regulation of ArfGAP3 activity. Our findings indicate that the two types of ArfGAP proteins that reside at the Golgi use a different combination of protein–protein and protein–lipid interactions to promote GTP hydrolysis in Arf1-GTP.

COPI vesicles accumulating in the presence of a GTP restricted arf1 mutant are depleted of anterograde and retrograde cargo

2000 ◽  
Vol 113 (1) ◽  
pp. 135-144 ◽  
Author(s):  
R. Pepperkok ◽  
J.A. Whitney ◽  
M. Gomez ◽  
T.E. Kreis
Keyword(s):  
Protein Transport ◽  
Ectopic Expression ◽  
Small Gtpase ◽  
Coated Vesicles ◽  
Gtp Hydrolysis ◽  
Rapid Accumulation ◽  
Gamma S

Microinjection of the slowly hydrolyzable GTP analogue GTP(gamma)S or the ectopic expression of a GTP restricted mutant of the small GTPase arf1 (arf1[Q71L]) leads to the rapid accumulation of COPI coated vesicles and buds in living cells. This effect is blocked at 15 degrees C and by microinjection of antibodies against (beta)-COP. Anterograde and retrograde membrane protein transport markers, which have been previously shown to be incorporated into COPI vesicles between the endoplasmic reticulum and Golgi complex, are depleted from the GTP(gamma)S or arf1[Q71L] induced COPI coated vesicles and buds. In contrast, in control cells 30 to 60% of the COPI carriers co-localize with these markers. These in vivo data corroborate recent in vitro work, suggesting that GTP(gamma)S and arf1[Q71L] interfere with the sorting of membrane proteins into Golgi derived COPI vesicles, and provide the first in vivo evidence for a role of GTP hydrolysis by arf1 in the sorting of cargo into COPI coated vesicles and buds.

scholarly journals EB3-informed dynamics of the microtubule stabilizing cap during stalled growth

2021 ◽  
Author(s):  
Maurits Kok ◽  
Florian Huber ◽  
Svenja-Marei Kalisch ◽  
Marileen Dogterom
Keyword(s):  
Growth Velocity ◽  
Hydrolysis Rate ◽  
Exact Relation ◽  
Gtp Hydrolysis ◽  
Lifetime Distributions ◽  
Microtubule Stability ◽  
In Vitro Reconstitution ◽  
1D Model ◽  
Microtubule Growth

Microtubule stability is known to be governed by a stabilizing GTP/GDP-Pi cap, but the exact relation between growth velocity, GTP hydrolysis and catastrophes remains unclear. We investigate the dynamics of the stabilizing cap through in vitro reconstitution of microtubule dynamics in contact with micro-fabricated barriers, using the plus-end binding protein GFP-EB3 as a marker for the nucleotide state of the tip. The interaction of growing microtubules with steric objects is known to slow down microtubule growth and accelerate catastrophes. We show that the lifetime distributions of stalled microtubules, as well as the corresponding lifetime distributions of freely growing microtubules, can be fully described with a simple phenomenological 1D model based on noisy microtubule growth and a single EB3-dependent hydrolysis rate. This same model is furthermore capable of explaining both the previously reported mild catastrophe dependence on microtubule growth rates and the catastrophe statistics during tubulin washout experiments.

scholarly journals Inhibition of GTP hydrolysis by Sar1p causes accumulation of vesicles that are a functional intermediate of the ER-to-Golgi transport in yeast

1994 ◽  
Vol 124 (4) ◽  
pp. 425-434 ◽  
Author(s):  
T Oka ◽  
A Nakano
Keyword(s):  
Protein Transport ◽  
Active Form ◽  
Integral Membrane Protein ◽  
Temperature Sensitive ◽  
Vesicle Formation ◽  
Gtp Hydrolysis ◽  
Golgi Transport ◽  
Transport Vesicles ◽  
In Vitro Reconstitution

The SAR1 gene product (Sar1p), a 21-kD GTPase, is a key component of the ER-to-Golgi transport in the budding yeast. We previously reported that the in vitro reconstitution of protein transport from the ER to the Golgi was dependent on Sar1p and Sec12p (Oka, T., S. Nishikawa, and A. Nakano. 1991. J. Cell Biol. 114:671-679). Sec12p is an integral membrane protein in the ER and is essential for the Sar1 function. In this paper, we show that Sar1p can remedy the temperature-sensitive defect of the sec12 mutant membranes, which is in the formation of ER-to-Golgi transport vesicles. The addition of Sar1p promotes vesicle formation from the ER irrespective of the GTP- or GTP gamma S-bound form, indicating that the active form of Sar1p but not the hydrolysis of GTP is required for this process. The inhibition of GTP hydrolysis blocks transport of vesicles to the Golgi and thus causes their accumulation. The accumulating vesicles, which carry Sar1p on them, can be separated from other membranes, and, after an appropriate wash that removes Sar1p, are capable of delivering the content to the Golgi when added back to fresh membranes. Thus we have established a new method for isolation of functional intermediate vesicles in the ER-to-Golgi transport. The sec23 mutant is defective in activation of Sar1 GTPase (Yoshihisa, T., C. Barlowe, and R. Schekman. 1993. Science (Wash. DC). 259:1466-1468). The membranes and cytosol from the sec23 mutant show only a partial defect in vesicle formation and this defect is also suppressed by the increase of Sar1p. Again GTP hydrolysis is not needed for the suppression of the defect in vesicle formation. Based on these results, we propose a model in which Sar1p in the GTP-bound form is required for the formation of transport vesicles from the ER and the GTP hydrolysis by Sar1p is essential for entering the next step of vesicular transport to the Golgi apparatus.

scholarly journals ADP-ribosylation Factor 1-independent Protein Sorting and Export from thetrans-Golgi Network

2004 ◽  
Vol 279 (50) ◽  
pp. 52735-52743 ◽  
Author(s):  
Mark A. Ellis ◽  
Mark T. Miedel ◽  
Christopher J. Guerriero ◽  
Ora A. Weisz
Keyword(s):  
Small Gtpase ◽  
Proton Channel ◽  
Coat Proteins ◽  
Adp Ribosylation ◽  
Influenza Hemagglutinin ◽  
Intact Membrane ◽  
Golgi Network ◽  
Adp Ribosylation Factor

Polarized epithelial cells efficiently sort newly synthesized apical and basolateral proteins into distinct transport carriers that emerge from thetrans-Golgi network (TGN), and this sorting is recapitulated in nonpolarized cells. While the targeting signals of basolaterally destined proteins are generally cytoplasmically disposed, apical sorting signals are not typically accessible to the cytosol, and the transport machinery required for segregation and export of apical cargo remains largely unknown. Here we investigated the molecular requirements for TGN export of the apical marker influenza hemagglutinin (HA) in HeLa cells using anin vitroreconstitution assay. HA was released from the TGN in intact membrane-bound compartments, and export was dependent on addition of an ATP-regenerating system and exogenous cytosol. HA release was inhibited by guanosine 5′-O-(3-thiotriphosphate) (GTPγS) as well as under conditions known to negatively regulate apical transportin vivo, including expression of the acid-activated proton channel influenza M2. Interestingly, release of HA was unaffected by depletion of ADP-ribosylation factor 1, a small GTPase that has been implicated in the recruitment of all known adaptors and coat proteins to the Golgi complex. Furthermore, regulation of HA release by GTPγS or M2 expression was unaffected by cytosolic depletion of ADP-ribosylation factor 1, suggesting that HA sorting remains functionally intact in the absence of the small GTPase. These data suggest that TGN sorting and export of influenza HA does not require classical adaptors involved in the formation of other classes of exocytic carriers and thus appears to proceed via a novel mechanism.

scholarly journals Fibrinogen anchors for micropatterning of active proteins and subcellular receptor relocalisation

2020 ◽  
Author(s):  
Joseph L. Watson ◽  
Samya Aich ◽  
Benjamí Oller Salvia ◽  
Andrew A. Drabek ◽  
Stephen C. Blacklow ◽  
...  
Keyword(s):  
Molecular Motors ◽  
Cell Biology ◽  
Cell Shape ◽  
Control Cell ◽  
High Specificity ◽  
Transmembrane Receptors ◽  
Current Technology ◽  
In Vitro Reconstitution ◽  
General Method

AbstractProtein micropatterning allows proteins to be precisely deposited onto a substrate of choice, and is now routinely used in cell biology and in vitro reconstitution. However, a drawback of current technology is that micropatterning efficiency can be variable between proteins, and that proteins may lose activity on the micropatterns. Here, we describe a general method to enable micropatterning of virtually any protein at high specificity and homogeneity while maintaining its activity. Our method is based on an anchor that micropatterns well, Fibrinogen, which we functionalized to bind to common purification tags. This enhances micropatterning on various substrates, facilitates multiplexed micropatterning, and dramatically improves the on-pattern activity of fragile proteins like molecular motors. Furthermore, it enhances the micropatterning of hard to micropattern cells. Last, this method enables subcellular micropatterning, whereby complex micropatterns simultaneously control cell shape and the distribution of transmembrane receptors within that cell. Altogether, these results open new avenues for cell biology.

scholarly journals High-efficacy subcellular micropatterning of proteins using fibrinogen anchors

2021 ◽  
Vol 220 (2) ◽  
Author(s):  
Joseph L. Watson ◽  
Samya Aich ◽  
Benjamí Oller-Salvia ◽  
Andrew A. Drabek ◽  
Stephen C. Blacklow ◽  
...  
Keyword(s):  
Molecular Motors ◽  
Cell Biology ◽  
Cell Shape ◽  
Control Cell ◽  
High Specificity ◽  
High Efficacy ◽  
Transmembrane Receptors ◽  
In Vitro Reconstitution ◽  
General Method

Protein micropatterning allows proteins to be precisely deposited onto a substrate of choice and is now routinely used in cell biology and in vitro reconstitution. However, drawbacks of current technology are that micropatterning efficiency can be variable between proteins and that proteins may lose activity on the micropatterns. Here, we describe a general method to enable micropatterning of virtually any protein at high specificity and homogeneity while maintaining its activity. Our method is based on an anchor that micropatterns well, fibrinogen, which we functionalized to bind to common purification tags. This enhances micropatterning on various substrates, facilitates multiplexed micropatterning, and dramatically improves the on-pattern activity of fragile proteins like molecular motors. Furthermore, it enhances the micropatterning of hard-to-micropattern cells. Last, this method enables subcellular micropatterning, whereby complex micropatterns simultaneously control cell shape and the distribution of transmembrane receptors within that cell. Altogether, these results open new avenues for cell biology.

scholarly journals Crucial Role of the Small GTPase ARF6 in Hepatic Cord Formation during Liver Development

2006 ◽  
Vol 26 (16) ◽  
pp. 6149-6156 ◽  
Author(s):  
Teruhiko Suzuki ◽  
Yoshiakira Kanai ◽  
Takahiko Hara ◽  
Junko Sasaki ◽  
Takehiko Sasaki ◽  
...  
Keyword(s):  
Collagen Gel ◽  
Small Gtpase ◽  
Liver Development ◽  
Phosphoinositide Metabolism ◽  
Fetal Hepatocytes ◽  
Cellular Events ◽  
Epithelial Sheet ◽  
Adp Ribosylation Factor

ABSTRACT The mammalian small GTPase ADP-ribosylation factor 6 (ARF6) plays important roles in a wide variety of cellular events, including endocytosis, actin cytoskeletal reorganization, and phosphoinositide metabolism. However, physiological functions for ARF6 have not previously been examined. Here, we described the consequence of ARF6 ablation in mice, which manifests most obviously in the context of liver development. Livers from ARF6 −/− embryos are smaller and exhibit hypocellularity, due to the onset of midgestational liver cell apoptosis. Preceding the apoptosis, however, defective hepatic cord formation is observed; the liver cells migrate abnormally upon exiting the primordial hepatic epithelial sheet and clump rather than becoming dispersed. Consistent with this observation, the ability of hepatocyte growth factor/scatter factor (HGF) to induce hepatic cord-like structures from ARF6 −/− fetal hepatocytes cultured in vitro in collagen gel matrix is impaired. Finally, we show that endogenous ARF6 in wild-type fetal hepatocytes is activated in response to HGF stimulation. These results provide evidence that ARF6 is an essential component in the signaling pathway coupling HGF signaling to hepatic cord formation.

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