@article {152168, title = {Motor Function of the Two-Component EEA1-Rab5 Revealed by dcFCCS.}, journal = {Methods Mol Biol}, volume = {2881}, year = {2025}, month = {2025}, pages = {87-115}, abstract = {

Fluorescence correlation spectroscopy (FCS) enables the measurement of fluctuations at fast timescales (typically few nanoseconds) and with high spatial resolution (tens of nanometers). This single-molecule measurement has been used to characterize single-molecule transport and flexibility of polymers and biomolecules such as DNA and RNA. Here, we apply this technique as dual-color fluorescence cross-correlation spectroscopy (dcFCCS) to identify the motor function of the tethering protein EEA1 and the small GTPase Rab5 by probing the flexibility changes through end-monomer fluctuations.

}, keywords = {Humans, rab5 GTP-Binding Proteins, Single Molecule Imaging, Spectrometry, Fluorescence, Vesicular Transport Proteins}, issn = {1940-6029}, doi = {10.1007/978-1-0716-4280-1_4}, author = {Soler, Joan Antoni and Singh, Anupam and Zerial, Marino and Thutupalli, Shashi} } @article {130535, title = {Two-component molecular motor driven by a GTPase cycle}, journal = {NATURE PHYSICS}, year = {2023}, month = {05/2023}, abstract = {

ATPases can cyclically convert free energy into mechanical work. Now, it is shown that the GTPase Rab5 can also perform mechanical work as part of a two-component molecular motor with the tethering protein EEA1.

ATPases are a group of enzymes that can cyclically convert the free energy of ATP hydrolysis into mechanical work. GTPases are another class of enzymes that are predominantly associated with signal transduction processes, but their role in mechanotransduction is less established. It was previously shown that the binding of the GTPase Rab5 to the tethering protein EEA1 induces a large conformational change in EEA1 from a rigid, extended to a flexible, collapsed state. This entropic collapse of EEA1 gives rise to an effective force that can pull tethered membranes closer. It currently remains unclear if EEA1 can return from the collapsed to the extended conformation without the aid of chaperone proteins. Here we show that EEA1 in a bulk solution can undergo multiple flexibility transition cycles driven by the energetics of Rab5 binding and unbinding as well as GTP hydrolysis. Each cycle can perform up to 20k(B)T of mechanical work. Hence, Rab5 and EEA1 constitute a two-component molecular motor driven by the chemical energy derived from the Rab5 GTPase cycle. We conclude that tethering proteins and their small GTPase partners can have active mechanical roles in membrane trafficking.

}, doi = {10.1038/s41567-023-02009-3}, author = {Singh, Anupam and Soler, Joan Antoni and Lauer, Janelle and Grill, Stephan W. and Jahnel, Marcus and Zerial, Marino and Thutupalli, Shashi} } @article {111547, title = {Active APPL1 sequestration by Plasmodium favors liver-stage development.}, journal = {Cell Rep}, volume = {39}, year = {2022}, month = {2022 May 31}, pages = {110886}, abstract = {

Intracellular pathogens manipulate host cells to survive and thrive. Cellular sensing and signaling pathways are among the key host machineries deregulated to favor infection. In this study, we show that liver-stage Plasmodium parasites compete with the host to sequester a host endosomal-adaptor protein (APPL1) known to regulate signaling in response to endocytosis. The enrichment of APPL1 at the parasitophorous vacuole membrane (PVM) involves an atypical Plasmodium Rab5 isoform (Rab5b). Depletion of host APPL1 alters neither the infection nor parasite development; however, upon overexpression of a GTPase-deficient host Rab5 mutant (hRab5_Q79L), the parasites are smaller and their PVM is stripped of APPL1. Infection with the GTPase-deficient Plasmodium berghei Rab5b mutant (PbRab5b_Q91L) in this case rescues the PVM APPL1 signal and parasite size. In summary, we observe a robust correlation between the level of APPL1 retention at the PVM and parasite size during exoerythrocytic development.

}, issn = {2211-1247}, doi = {10.1016/j.celrep.2022.110886}, author = {Lahree, Aparajita and Baptista, Sara de Jesus Santos and Marques, Sofia and Perschin, Veronika and Zuzarte-Lu{\'\i}s, Vanessa and Goel, Manisha and Choudhary, Hadi Hasan and Mishra, Satish and Stigloher, Christian and Zerial, Marino and Sundaramurthy, Varadharajan and Mota, Maria M} }