Dynamic Changes in the Spatiotemporal Localization of Rab21 in Live RAW264 Cells during Macropinocytosis

Rab21, a member of the Rab GTPase family, is known to be involved in membrane trafficking, but its implication in macropinocytosis is unclear. We analyzed the spatiotemporal localization of Rab21 in M-CSF-stimulated RAW264 macrophages by the live-cell imaging of fluorescent protein-fused Rab21. It was demonstrated that wild-type Rab21 was transiently associated with macropinosomes. Rab21 was recruited to the macropinosomes after a decrease in PI(4,5)P2 and PI(3,4,5)P3 levels. Although Rab21 was largely colocalized with Rab5, the recruitment of Rab21 to the macropinosomes lagged a minute behind that of Rab5, and preceded that of Rab7. Then, Rab21 was dissociated from the macropinosomes prior to the accumulation of Lamp1, a late endosomal/lysosomal marker. Our analysis of Rab21 mutants revealed that the GTP-bound mutant, Rab21-Q78L, was recruited to the macropinosomes, similarly to wild-type Rab21. However, the GDP-bound mutant, Rab21-T33N, did not localize on the formed macropinosomes, suggesting that the binding of GTP to Rab21 is required for the proper recruitment of Rab21 onto the macropinosomes. However, neither mutation of Rab21 significantly affected the rate of macropinosome formation. These data indicate that Rab21 is a transient component of early and intermediate stages of macropinocytosis, and probably functions in macropinosome maturation before fusing with lysosomal compartments

Comprehensive Analysis Reveals Dynamic and Evolutionary Plasticity of Rab GTPases and Membrane Traffic in Tetrahymena thermophila

Cellular sophistication is not exclusive to multicellular organisms, and unicellular eukaryotes can resemble differentiated animal cells in their complex network of membrane-bound structures. These comparisons can be illuminated by genome-wide surveys of key gene families. We report a systematic analysis of Rabs in a complex unicellular Ciliate, including gene prediction and phylogenetic clustering, expression profiling based on public data, and Green Fluorescent Protein (GFP) tagging. Rabs are monomeric GTPases that regulate membrane traffic. Because Rabs act as compartment-specific determinants, the number of Rabs in an organism reflects intracellular complexity. The Tetrahymena Rab family is similar in size to that in humans and includes both expansions in conserved Rab clades as well as many divergent Rabs. Importantly, more than 90% of Rabs are expressed concurrently in growing cells, while only a small subset appears specialized for other conditions. By localizing most Rabs in living cells, we could assign the majority to specific compartments. These results validated most phylogenetic assignments, but also indicated that some sequence-conserved Rabs were co-opted for novel functions. Our survey uncovered a rare example of a nuclear Rab and substantiated the existence of a previously unrecognized core Rab clade in eukaryotes. Strikingly, several functionally conserved pathways or structures were found to be associated entirely with divergent Rabs. These pathways may have permitted rapid evolution of the associated Rabs or may have arisen independently in diverse lineages and then converged. Thus, characterizing entire gene families can provide insight into the evolutionary flexibility of fundamental cellular pathways

Disease: A Hitherto Unexplored Constraint on the Spread of Dogs (Canis lupus familiaris) in Pre-Columbian South America

Although debate continues, there is agreement that dogs (Canis lupus familiaris) were first domesticated in Eurasia, spreading from there to other parts of the world. However, while that expansion already extended as far as Europe, China, and North America by the early Holocene, dogs spread into (and south of) the tropics only much later. In South America, for example, the earliest well attested instances of their presence do not reach back much beyond 3000 cal. BC, and dogs were still absent from large parts of the continent – Amazonia, the Gran Chaco, and much of the Southern Cone – at European contact. Previous explanations for these patterns have focused on cultural choice, the unsuitability of dogs for hunting certain kinds of tropical forest prey, and otherwise unspecified environmental hazards, while acknowledging that Neotropical lowland forests witness high rates of canine mortality. Building on previous work in Sub-Saharan Africa (Mitchell 2015) and noting that the dog’s closest relatives, the grey wolf (C. lupus) and the coyote (C. latrans), were likewise absent from South and most of Central America in Pre- Columbian times, this paper explores instead the possibility that infectious disease constrained the spread of dogs into Neotropical environments. Four diseases are considered, all likely to be native and/or endemic to South America: canine distemper, canine trypanosomiasis, canine rangeliosis, and canine visceral leishmaniasis caused by infection with Leishmania amazonensis and L. colombiensis. The paper concludes by suggesting ways in which the hypothesis that disease constrained the expansion of dogs into South America can be developed further

Constant hepatic ATP concentrations during prolonged fasting and absence of effects of Cerbomed Nemos(®) on parasympathetic tone and hepatic energy metabolism.

OBJECTIVE: Brain insulin-induced improvement in glucose homeostasis has been proposed to be mediated by the parasympathetic nervous system. Non-invasive transcutaneous auricular vagus nerve stimulation (taVNS) activating afferent branches of the vagus nerve may prevent hyperglycemia in diabetes models. We examined the effects of 14-min taVNS vs sham stimulation by Cerbomed Nemos(®) on glucose metabolism, lipids, and hepatic energy homeostasis in fasted healthy humans (n = 10, age 51 ± 6 yrs, BMI 25.5 ± 2.7 kg/m(2)). METHODS: Heart rate variability (HRV), reflecting sympathetic and parasympathetic nerve activity, was measured before, during and after taVNS or sham stimulation. Endogenous glucose production was determined using [6,6-(2)H2]glucose, and hepatic concentrations of triglycerides (HCL), adenosine triphosphate (ATP), and inorganic phosphate (Pi) were quantified from (1)H/(31)P magnetic resonance spectroscopy at baseline and for 180 min following stimulation. RESULTS: taVNS did not affect circulating glucose, free fatty acids, insulin, glucagon, or pancreatic polypeptide. Rates of endogenous glucose production (P = 0.79), hepatic HCL, ATP, and Pi were also not different (P = 0.91, P = 0.48 and P = 0.24) between taVNS or sham stimulation. Hepatic HCL, ATP, and Pi remained constant during prolonged fasting for 3 h. No changes in heart rate or shift in cardiac autonomic function from HRV towards sympathetic or parasympathetic predominance were detected. CONCLUSION: Non-invasive vagus stimulation by Cerbomed Nemos(®) does not acutely modulate the autonomic tone to the visceral organs and thereby does not affect hepatic glucose and energy metabolism. This technique is therefore unable to mimic brain insulin-mediated effects on peripheral homeostasis in humans

Rab GTPases act in sequential steps to regulate phagolysosome formation

During apoptosis, apoptotic cells are recognized and quickly engulfed by phagocytes. The internalized cell corpses are enclosed within membrane-bound vesicles called phagosomes. Cell corpse degradation depends on the phagosomes undergoing a maturation process, but regulation of phagosomal maturation is not well understood. Recently, we identified C. elegans Rab GTPase 14 as a novel regulator of apoptotic cell degradation. Loss of rab-14 function affects several steps of phagosome maturation, causing accumulation of persistent cell corpses. RAB-14 and UNC-108 (Rab GTPase 2) function redundantly to regulate phagosome maturation. Three Rabs, RAB-14, UNC-108/RAB2 and RAB-7, act cooperatively to control phagolysosome formation. RAB-14 and UNC-108 recruit lysosomes, while RAB-7 mediates fusion of lysosomes to phagosomes. Our data thus reveal the sequential action of Rab GTPases in regulating tethering, docking and fusion of lysosomes to phagosomes