Rab3a and Rab10 are regulators of lysosome exocytosis and plasma membrane repair

ABSTRACT

Disruption of the cell plasma membrane can occur due to mechanical damage, pore forming toxins, etc. Resealing or plasma membrane repair (PMR) is the emergency response required for cell survival. It is triggered by Ca²⁺ entering through the disruption, causing organelles such as lysosomes located underneath the plasma membrane to fuse rapidly with the adjacent plasma membrane. We have recently identified some of the molecular traffic machinery that is involved in this vital process. Specifically, we showed that 2 members of the Rab family of small GTPases, Rab3a and Rab10, are essential for lysosome exocytosis and PMR in cells challenged with a bacterial toxin, streptolysin-O (SLO). Additionally, we showed that Rab3a regulates PMR via the interaction with 2 effectors, synaptotagmin-like protein 4a (Slp4-a) and nonmuscle myosin heavy chain IIA (NMHC IIA), the latter being identified for the first time as a Rab3a effector. This tripartite complex is essential for the positioning of the peripheral lysosomes responsible for PMR. In cells lacking any of the components of this tripartite complex, lysosomes were concentrated in the perinuclear region and absent in the periphery culminating with PMR inhibition.

When the plasma membrane loses its integrity, repair has to occur within seconds to avoid toxic influxes of extracellular molecules and ions and effluxes of intracellular components—both processes leading to cell death. Plasma membrane repair (PMR) is composed of 2 main processes: Ca²⁺ influx, proposed to be the initial signal for PMR, followed by rapid exocytosis of endocytic vesicles that are then responsible for membrane resealing.^1,2 The source of the membrane vesicles that fuse with the damaged plasma membrane (PM) in response to the Ca²⁺ signal are lysosomes located at the periphery of the cell near the PM.^2,3 Pairing of N-ethylmaleimide sensitive factor attachment protein receptors (SNARES), located at the lysosomal and plasma membrane, with each other allows fusion of the peripheral lysosomes with the PM. Increase in the cytosolic Ca²⁺ levels causes a conformational change in synaptotagmin VII, the Ca²⁺-sensing SNARE-interacting protein in the lysosome membrane, enabling its interaction with VAMP-7 in lysosomes and SNAP-23 and syntaxin-4 in the PM.⁴ For the first time, our recent publication⁵ sheds light on the Rab GTPases involved in lysosome exocytosis and in PMR by these organelles. The two processes share similar traffic machinery. Rab GTPases localize to specific subcellular compartments, contributing to organelle identity and directing organelle trafficking. It is thought that these proteins regulate specific pathways such as vesicle movement, tethering and fusion events through interactions with coat, motor, tether and SNARE proteins.^6,7

To identify Rab GTPases involved in PMR, we screened 58 Rabs, using a lentiviral shRNA library from the BROAD RNAi consortium. Surprisingly, this screen identified only 2 Rabs, Rab3a and Rab10, as being crucial for lysosome exocytosis and PMR.⁵ These results were completely unexpected since Rab3a had never before been linked to conventional lysosomes from non-specialized secretory cells. It is known that Rab3a localizes to synaptosomes and secretory granules, playing a critical role in regulated secretion and neurotransmitter release. Indeed, Rab3a and its isoforms (b, c and d) are the most extensively investigated Rabs in terms of regulated secretion.^8-10 Furthermore, Südhof and co-workers¹¹ have generated multiple knockouts for the Rab3 isoforms, and found that all single and double Rab3 knock-out mice were viable and fertile, suggesting redundancy among the 4 Rab3 isoforms.

Our screen was performed in non-secretory THP-1 and Hela cells. In the latter cell type, Rab3a is localized mainly in the Golgi but live confocal- and total internal reflection fluorescence- microscopy revealed that Rab3a is localized in a subset of lysosomes at the periphery of the cell. Interestingly, silencing of Rab3a resulted in collapse of the peripheral lysosomes to the perinuclear region and subsequent inhibition of PMR.⁵ This outcome was not observed for the other Rab3 isoforms expressed in HeLa cells. Rab GTPases control vesicular traffic by recruiting effector proteins that bind exclusively to the GTP-bound, active form of the GTPase.¹² Of the 4 Rab3a effectors tested in our publication, all well described in regulated secretion, only the silencing of synaptotagmin-like protein 4a (Slp4-a) was able to phenocopy the Rab3a-silenced phenotype. We were also able to identify a new Rab3a-effector, an actin-dependent motor adaptor, nonmuscle myosin heavy chain IIA (NMHC IIA) that is also critical for lysosome positioning at the periphery of the cell and for the exocytosis of these organelles.⁵ NMHC IIA has primarily been described to be involved in tension-related cellular processes but this myosin has also been found to facilitate vesicle trafficking required for PM repair in other experimental settings.¹³

The identification of Rab3a and its effectors Slp4-a and NMHC IIA as regulators of lysosome positioning and PM repair represents a significant advance in our understanding of how lysosomes stay at the pheriphery of the cell and their recruitment to repair damaged PM. The exact functions of the proteins of this tripartite complex and the sequential manner in which they function remains unknown. Nevertheless, taking into account our achievements and the well-known role of Rab3a in regulated secretion we can state that Rab3a plays a key role in exocytosis in general and that the underlying mechanisms are dependent on the set of effectors recruited.

We also proposed a working model in which Rab3a is responsible for keeping the pool of lysosomes at the periphery of the cell by interacting with the cortical actin cytoskeleton via its effector NMHC IIA. The role of Slp4-a in the positioning of lysosomes at the cell periphery is still an open question, although we cannot exclude an interaction between NMHC IIA and Slp4-a (via its linker domain), because the silencing of the latter was sufficient to induce lysosome clustering.⁵

This working model has striking analogies with the models proposed for the function of Rab27a in regulated secretion of lysosome related organelles. Indeed, several studies indicate that Rab27a, Melanophilin, and MyoVa mediate the physical link between melanosomes and cortical actin.^14,15 In retinal pigment epithelial cells, a complex of Rab27a, MyRIP, and Myosin VIIA was also identified to have similar functions, since these cells also possess melanosomes.¹⁶ Interestingly, these tripartite complexes are composed by Rab27a, the closest Rab to the Rab3 subfamily by a protein that has similar functions and a common Rab-binding domain to Slp4-a (Melanophilin or MyRIP) and a Myosin.¹⁷

From the 60 Rab GTPases existent in the human genome very few have been associated with lysosomes. The finding that Rab3a is localized with peripheral lysosomes contributes to fill this gap. Rab7, has been described to localize in late endosomal system and is crucial in lysosome biogenesis.^18,19 More recently, attention has focused on the role of an Arl GTPase, Arl8b – the only known small GTP-binding protein that is present primarily on mature lysosomes.^20,21 This small GTPase has recently emerged as a crucial regulator of membrane trafficking toward lysosomes and lysosome positioning. BORC, a key component of the molecular machinery that regulates lysosome translocation to the periphery of the cell, recruits Arl8b to the lysosomal membrane enabling coupling to the SKIP–kinesin-1 complex that drives microtubule-guided movement of lysosomes toward the cell periphery.^22-24

The role of the other Rab protein/s required for lysosome exocytosis and PMR, Rab10, is/are not yet well established. Work from different groups has placed Rab10 within the endocytic/recycling system and no link between Rab10 and lysosomes has been reported so far.^25,26 In different cell types Rab10 localizes on both Golgi and early endosomal/recycling compartments but not on late endosomes or lysosomes.^25,26 Due to the robustness of Rab10 phenotypes in lysosome exocytosis and PMR we propose that peripheral lysosomes and recycling endosomes do interact. It will be very interesting to understand the mechanisms behind this interaction and how it affects lysosome exocytosis and PMR.

The new microscopy techniques for following lysosome biogenesis in real time and the ability to individually silence components of the trafficking machinery will certainly bring new insights in this area and will contribute to a more complete understanding of lysosome biogenesis, exocytosis and PMR.

Abbreviations

NMHC IIA

nonmuscle myosin heavy chain IIA

PM

Plasma membrane

PMR

Plasma membrane repair

SNARE

N-ethylmaleimide sensitive factor attachment protein receptors

SLO

Streptolysin-O

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

Supported by the Portuguese FCT [HMSP-ICT/0024/2010, co-funded by the EU (FEDER – Fundo Europeu de Desenvolvimento Regional) through COMPETE – Programa Operacional Factores de Competitividade and QREN – Quadro de Referência Estratégico], iNOVA4Health - UID/Multi/04462/2013.