Small GTPase networks

Complex biochemical networks control the activation of small GTPases in space and time.

The most important proteins organizing intracellular transport are small GTPases, molecular switches that can exist in an ON- and OFF-state regulating vesicle formation, transport and fusion. Importantly, the activities of these proteins are regulated in complex biochemical networks, which can give rise to emergent properties and collective behavior.

In this research project, we are using an in vitro reconstitution approach to understand the logic of the regulatory networks that control small GTPases.

Recently, we have used our in vitro reconstitution approach to answer the following questions:

What are the emergent properties of the regulatory network controlling Rab5 activation?
While the GTPase switch is well understood on a molecular level, the cellular function of small GTPases relies on their collective behavior, where large ensembles of the proteins switch simultaneously. Experimental and theoretical evidence indicated that this collective switching could be the result of complex biochemical circuits that include non-linear interactions such as positive feedback loops. However, the minimal biochemical system able to generate such behavior had so far not been identified. In this collaborative project with the group of Timothy Saunders, we combined in vitro reconstitution experiments with stochastic reaction-diffusion modeling to study the emergent properties of a minimal Rab5 GTPase activation network comprised of Rab5, its GDP dissociation inhibitor (GDI) and nucleotide exchange factor Rabex5, the scaffold protein Rabaptin5 and biomimetic membranes. We found that collective GTPase switching relies on a positive feedback and involves mechanisms that repress indiscriminate noise amplification. Furthermore, our experiments provide evidence that controlling the inactive population of Rab5 on the membrane can shape the network response. Notably, we demonstrate that collective switching can spread on the membrane surface as a traveling wave of Rab5 activation. Together, these findings reveal how biochemical networks control vectorial vesicle trafficking pathways and how their non-equilibrium properties define the spatiotemporal organization of the cell (Bezeljak et al., 2020).

The Rab5 activation network gives rise to bistability and the coexistence of the ON- and OFF-state. As a result we can observe traveling waves of Rab5 activation on the membrane. Importantly, such spatial patterns share features with activity patterns found for Rab5 in vivo.

What controls the activity of Arf6 in a nascent neuron?
Eukaryotic cell differentiation 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 has been implicated in the control of the small GTPase Arf6 and dendrite branching. It is also thought to mediate axon specific delivery of PI(3,4,5)P₃ together with the motor protein KIF13B, thus contributing to phosphoinositide polarity (Hansen et al., 2017). However, how these different roles of ADAP1 are coordinated remains unclear. We studied ADAP1’s functions using in vitro reconstitutions and found that KIF13B transports ADAP1 along microtubules. Instead of being a cargo, as previously believed, we found that PIP₃ instead acts as a stop signal for this transport. At the same time, PIP₃ and PI(3,4)P₂ activate ADAP1s enzymatic activity to catalyze GTP hydrolysis by Arf6. Our results support a model, by which KIF13B transports ADAP1 towards high concentrations of PIP₃/PI(3,4)P₂ in the plasma membrane, where it disassociates from the motor and inactivates Arf6. Importantly, these findings demonstrate that phosphoinositides can act as cargo-release factors and challenge current models for how polarity is established in neurons (Duellberg & Auer et al., 2021).

Proposed model for the action of ADAP1. KIF13B transports ADAP1 along microtubules in dendrites (i) until it reaches regions of high PIP3 or PI(3,4)P2 concentration. Here, ADAP1 is released from KIF13B (ii) and recruited to the membrane (iii), where it stimulates GTP hydrolysis and membrane detachment of ARF6 (iv).

PEOPLE WORKING ON THIS PROJECT

Albert, Lukasz, Gabriel, Zane

RELEVANT PUBLICATIONS

Christian Duellberg, Albert Auer, Nicola Canigova, Katrin Loibl, Martin Loose.
In vitro reconstitution reveals phosphoinositides as cargo-release factors and activators of the ARF6 GAP ADAP1
PNAS (2021), vol. 118 (1) e2010054118.

U. Bezeljak, H. Loya, B. Kaczmarek, T. E. Saunders and M. Loose.
Stochastic activation and bistability in a Rab GTPase regulatory network .
PNAS (2020), vol. 117(12):6540-6549.

P. A. Nguyen*, A. C. Groen*, M. Loose, K. Ishihara, M. Wühr, C. M. Field, and T. J. Mitchison.
Spatial Organization of Cytokinesis Signaling Reconstituted in a Cell-Free System.
Science (2014) vol. 346, 244-247

FUNDING

This research project has received funding from the Human Frontiers Science Program (HFSP), Grant No. RGY0083/2016,