| dc.description.abstract | The endomembrane system and their associated membrane trafficking are fundamental
features of eukaryotic cell biology. These processes are regulated by small GTP-binding
proteins that acts as molecular switches to control signalling based on the type of bound
nucleotide. Small GTPases function to exert spatial and temporal regulation on intracellular
trafficking. Amongst the Ras superfamily of small GTPases, the Rab subfamily consists of
around 70 members. Rabs serve as markers for membrane/organelle identity and function as
regulatory factors in vesicle transport. In this study, we attempted to characterize the
endosomal function of the small GTPase Rab14.
Rab14 was identified as a low molecular weight GTP-binding protein (Elferink et al., 1992).
This protein localizes to trans-Golgi, early and recycling endosomes and has been shown to
control the transport from the Golgi and endosomes. Rab14 has been implicated in multiple
cellular processes including development (Ueno et al., 2011) and immune response. Multiple
studies have described the mechanism of Rab14 function in endosomal recycling. Here, we
attempted to investigate its function in the biogenesis of tubular recycling endosomes (TREs).
Objective : Studying the regulation of recycling endosomes by Rab14
Rab14 has been shown to localize to endosomal compartments in addition to trans-Golgi
(Junutula et al., 2004). Although it is involved in endocytic recycling, its precise role in
regulating the recycling of cargo such as transferrin has not been completely understood.
Expression of GFP-Rab14 in HeLa cells showed its localization to early endosomes (EEA1)
and recycling endosomes (TfR). Further, constitutive active mutant of GFP-Rab14 also
showed its localization to these compartments. However, the dominant negative mutant of
GFP-Rab14 did not show its effect as expected but localized to trans-Golgi as shown by
other groups. Multiple Rabs are known to localize to endosomal compartments. However,
whether and how Rab14 regulates other endosomal structures labelled by Rabs have not been
well studied. To study the effect of Rab14 on endo-lysosomal pathway, we co-expressed
GFP-Rab14WT and its mutants with mCherry-tagged Rab11A or Rab22A to represent REs
and Rab7A to mark late endosomes/lysosomes. Our studies show that Rab14 co-localizes
highly with RE localized Rabs Rab11A and Rab22A, and to a lesser extent with Rab7A.
Consistently, overexpression of constitutive active mutant of Rab14 causes increased co localization with co-expressed Rabs. To study the possible regulation of Rab14 on recycling
endosomes, we employed a siRNA-mediated knockdown approach. Depletion of Rab14
levels did not significantly affect the length or the number of tubules labelled by the kinesin-3
motor KIF13A or the small GTPase Rab22A. We also checked the status of the CIE cargo,
CD147, which traffics through tubular recycling endosomes. However, we noticed that
Rab14 knockdown significantly decreased the percentage of cells showing endogenous
Rab11A tubules. Interestingly, we also observed that Rab11A staining of Rab14-depleted
cells under methanol fixation conditions displayed enlarged Rab11A vesicular structures,
which was not observed in control cells.
Rab11A tubules can carry both CIE as well as CME cargo. To confirm if Rab11A-labelled
tubules were broadly affected, we also assessed the status of CIE marker CD55 as well as
CD147 in Rab14-depleted conditions. To study if Rab11A knockdown can affect Rab14
localization or Rab14 compartments, we knocked down Rab11A and expressed GFP-tagged
Rab14. However, we did not observe any significant effect of Rab11A knockdown on Rab14.
These observations suggest that Rab14 possibly may work upstream and/or regulate a subset
of Rab11A-positive tubules. For recycling of cargo to the plasma membrane, endosomal
tubules are either generated from early endosomes or from the endocytic recycling
compartment (ERC). To investigate which of these two spatially distributed REs are
regulated by Rab14, we stained Rab14 knockdown cells with a combination of cargo (marked
by TfR)/organelle markers to study if Rab14 regulates Rab11A-dependent cargo localization
and transport from early endosomes to recycling endosomes or recycling towards the plasma
membrane. Our results show that Rab14 does not significantly affect cargo localization and
transport to and from Rab11A endosomes, indicating the possibility of alternate redundant or
parallel pathways to ameliorate block in the Rab14-dependent recycling pathway.
We also tested if Rab14 levels may affect late endosomal-lysosomal compartments using
markers specific for late endosomal and lysosomal markers. Our results do not show
significant changes in endo-lysosomes suggesting that Rab14 is primarily associated with
early and recycling endosomes and may not directly be involved in regulating late
endosomal-lysosomal compartments.
A number of molecules have been shown in literature to regulate Rab11A tubules. Based on
literature and our observations, we hypothesized that Rab14 may recruit sorting nexins,
probably SNX4 or SNX1 (which are known membrane sculpting and remodelling
molecules), to modulate Rab11A endosome tubules downstream. We overexpressed SNX4
and SNX1 in Rab14 depleted cells and made observations on its cellular distribution and
localization. Our results suggest that Rab14 may work through different mechanisms or
effectors to modulate Rab11A tubules. Overall, our results suggest that Rab14 acts upstream
of Rab11A and regulates Rab11A endosomal compartments or a subset of Rab11A
structures. | en_US |
