There and back again: hair follicle stem cell dynamics

Recently in Cell, Hsu et al. (2011) defined the relationship between stem cells and differentiated progeny within a hair follicle lineage. Their work reveals that stem cell descendants that have migrated out of the bulge can return to this niche and actively contribute to its function.     

Intracellular trafficking of recycling apolipoprotein E in Chinese hamster ovary cells

We have investigated apolipoprotein E (apoE) recycling in Chinese hamster ovary (CHO) cells, a peripheral cell that does not produce lipoproteins or express apoE. Using a pulse-chase protocol in which cells were pulsed with 125I-apoE-VLDL and chased for different periods, approximately 30% of the apoE internalized during the pulse was resecreted within a 4 h chase in a relatively lipid-free state. The addition of lysosomotropic agents or brefeldin A had no effect on apoE recycling. Unlike previous results with hepatocytes and macrophages, neither apoA-I nor upregulation of ABCA1 stimulated apoE recycling. However, cyclodextrin, which extracts cholesterol from plasma membrane lipid rafts, increased recycling. Confocal studies revealed that apoE, internalized during a 1 h pulse, colocalizes with early endosomal antigen-1, Rab5, Rab11a, and lysobisphosphatidic acid but not with lysosomal-associated membrane protein-1. Colocalization of apoE and Rab11a persisted even after cells had been chased for 1 h, suggesting a pool of apoE within the endosomal recycling compartment (ERC). Our data suggest that apoE recycling in CHO cells is linked to cellular cholesterol removal via the ERC and phospholipid-containing acceptors in a pathway alternative to the ABCA1-apoA-I axis.     

There and (slowly) back again: entropy-driven hysteresis in a model of DNA overstretching

When pulled along its axis, double-stranded DNA elongates abruptly at a force of ∼65 pN. Two physical pictures have been developed to describe this overstretched state. The first proposes that strong forces induce a phase transition to a molten state consisting of unhybridized single strands. The second picture introduces an elongated hybridized phase called S-DNA. Little thermodynamic evidence exists to discriminate directly between these competing pictures. Here we show that within a microscopic model of DNA we can distinguish between the dynamics associated with each. In experiment, considerable hysteresis in a cycle of stretching and shortening develops as temperature is increased. Since there are few possible causes of hysteresis in a system whose extent is appreciable in only one dimension, such behavior offers a discriminating test of the two pictures of overstretching. Most experiments are performed upon nicked DNA, permitting the detachment (unpeeling) of strands. We show that the long-wavelength progression of the unpeeled front generates hysteresis, the character of which agrees with experiment only if we assume the existence of S-DNA. We also show that internal melting can generate hysteresis, the degree of which depends upon the nonextensive loop entropy of single-stranded DNA.     

There and back again: switching between host orders by avian body lice (Ischnocera: Goniodidae)

Studies of major switches by parasites between highly divergent host lineages are important for understanding new opportunities for parasite diversification. One such major host switch is inferred for avian feather lice (Ischnocera) in the family Goniodidae, which parasitize two distantly‐related groups of birds: Galliformes (pheasants, quail, partridges, etc.) and Columbiformes (pigeons and doves). Although there have been several cophylogenetic studies of lice at the species level, few studies have focused on such broad evolutionary patterns and major host‐switching events. Using a phylogeny based on DNA sequences for goniodid feather lice, we investigated the direction of this major host switch. Unexpectedly, we found that goniodid feather lice have switched host orders, not just once, but twice. A primary host switch occurred from Galliformes to Columbiformes, leading to a large radiation of columbiform body lice. Subsequently, there was also a host switch from Columbiformes back to Galliformes, specifically to megapodes in the Papua–Australasian region. The results of the present study further reveal that, although morphologically diagnosable lineages are supported by molecular data, many of the existing genera are not monophyletic and a revision of generic limits is needed. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 102 , 614–625.     

Polymorphonuclear leukocytes promote neurotoxicity through release of matrix metalloproteinases, reactive oxygen species, and TNF-alpha

As the first immune cells to infiltrate the nervous system after traumatic PNS and CNS injury, neutrophils (polymorphonuclear leukocytes, PMNs) may promote injury by releasing toxic soluble factors that may affect neuronal survival. Direct neurotoxicity of matrix metalloproteinases (MMPs), reactive oxygen species (ROS), and cytokines released by PMNs was investigated by culturing dorsal root ganglion (DRG) cells with PMN-conditioned media containing MMP inhibitor (GM6001), ROS scavengers, or tumor necrosis factor alphaR (TNF-alphaR) neutralizing antibody. Although DRGs exposed to PMN-conditioned media had 53% fewer surviving neurons than controls, neuronal cell loss was prevented by GM6001 (20 micromol/L), catalase (1000 U/mL), or TNF-alphaR neutralizing antibody (1.5 microg/mL), elevating survival to 77%, 94%, and 95%, respectively. In accordance with protection by GM6001, conditioned media collected from MMP-9 null PMNs was less neurotoxic than that collected from wild-type PMNs. Additionally, MMP inhibition reduced PMN-derived ROS; removal of ROS reduced PMN-derived MMP-9 activity; and TNF-alpha inhibition reduced both PMN-derived MMP-9 activity and ROS in PMN cultures. Our data provide the first direct evidence that PMN-driven neurotoxicity is dependent on MMPs, ROS, and TNF-alpha, and that these factors may regulate PMN release of these soluble factors or interact with one another to mediate PMN-driven neurotoxicity.     

Intracellular trafficking of P-glycoprotein

Overexpression of P-glycoprotein (P-gp) is a major cause of multidrug resistance in cancer. P-gp is mainly localized in the plasma membrane and can efflux structurally and chemically unrelated substrates, including anticancer drugs. P-gp is also localized in intracellular compartments, such as endoplasmic reticulum (ER), Golgi, endosomes and lysosomes, and cycles between endosomal compartments and the plasma membrane in a microtubular-actin dependent manner. Intracellular trafficking pathways for P-gp and participation of different Rab proteins depend on cellular polarization and choice of primary culture, cell line or neoplasm. Interruption of P-gp trafficking to the plasma membrane increases intracellular P-gp accumulation and anticancer drug levels, suggesting a potential approach to overcome P-gp-mediated multidrug resistance in cancer.     

Out of Africa and back again

If the genusHomo did indeed originate in Africa, then it must have spread by about 2 m.y. ago into Asia where it is represented at 1.8 m.y. ago byHomo erectus fossils. This latter species in turn eventually spread back into Africa, as indicated by the 1.4 m.y. old OH 9 calvaria from Olduvai, and into Europe, as indicated by the 800,000 year old Ceprano calvaria from Italy. These hominids are associated only with Oldowan style artefacts of cores, choppers and flakes and were apparently not conversant with Acheulean handaxe technology. It seems that they most probably evolved viaHomo heidelbergensis into the Neanderthals. Meanwhile, a completely separate development originating withHomo ergaster of about 1.7 m.y. ago in Africa and possessing Acheulean handaxe technology evolved via such forms as Ndutu and Steinheim intoHomo sapiens.     

Intracellular trafficking and activation of the furin proprotein convertase: localization to the TGN and recycling from the cell surface.

Furin is a membrane-associated endoprotease that efficiently cleaves precursor proteins on the C-terminal side of the consensus sequence, Arg-X-Lys/Arg-Arg1, and has been proposed to catalyze these reactions in both exocytic and endocytic compartments. To study its biosynthesis and routing, a furin construct (designated fur/f) containing the FLAG epitope tag inserted on the C-terminal side of the enzyme's autoproteolytic maturation site was used. Introduction of the epitope tag had no effect on the expression, proteolytic maturation or activity of furin. Analysis of the localization of fur/f by immunofluorescence microscopy showed that its staining pattern largely overlapped with those of several Golgi-associated markers. Treatment of cells with brefeldin A caused the fur/f distribution to collapse around the microtubule organizing center, indicating that furin is concentrated in the trans-Golgi network (TGN). Immunoelectron microscopy showed unequivocally that furin resides in the TGN where it colocalized with TGN38. In agreement with its proposed activity in multiple compartments, antibody uptake studies showed that fur/f cycles between the cell surface and TGN. Furthermore, targeting to the TGN requires sequences in the cytoplasmic tail of the enzyme. Pulse-chase and immunofluorescence analyses demonstrated that proregion removal occurs in the endoplasmic reticulum and that cleavage may be required for exist from this compartment. Finally, we show that proregion removal is necessary but not sufficient for enzyme activation.     

Intracellular trafficking of Parachlamydia acanthamoebae

Parachlamydia acanthamoebae is an obligate intracellular bacterium that naturally infects free-living amoebae. It is a potential agent of pneumonia that resists destruction by human macrophages. However, the strategy used by this Chlamydia-like organism in order to resist to macrophage destruction is unknown. We analysed the intracellular trafficking of P. acanthamoebae within monocyte-derived macrophages. Infected cells were immunolabelled for the bacteria and for various intracellular compartments by using specific antibodies. We analysed the bacteria colocalization with the different subcellular compartments by using epifluorescence and confocal microscopy. Bacterial replication took place 4-6 h post infection within acidic vacuoles. At that time, P. acanthamoebae colocalized with Lamp-1, a membrane marker of late endosomal and lysosomal compartments. A transient accumulation of EEA1 15 min post infection, and of rab7 and the mannose 6-phosphate receptor 30 min post infection confirmed that P. acanthamoebae traffic through the endocytic pathway. The acquisition of Lamp-1 was not different after infection with living and heat-inactivated bacteria. However, 24.5% and 79.5% of living and heat-inactivated P. acanthamoebae, respectively, colocalized with the vacuolar proton ATPase. Moreover, P. acanthamoebae did not colocalized with cathepsin D, a lysosomal hydrolase, suggesting that P. acanthamoebae interferes with maturation of its vacuole. Thus, P. acanthamoebae survives to destruction by human macrophages probably by controlling the vacuole biogenesis.     

Intracellular trafficking of Shiga-toxin-B-subunit-functionalized spherulites

Background information. Spherulites are multi‐lamellar lipidic vesicles that can encapsulate biomolecules and may be used as carriers for drug delivery. STxB (Shiga toxin B‐subunit) is known to bind the glycosphingolipid Gb3 (globotriaosyl ceramide), which is overexpressed by various human tumours. After Gb3 binding, the toxin enters the cytoplasm via the retrograde route, bypassing the degrading environment of the late endosomes/lysosomes. STxB is non‐toxic and has been identified as a promising tool for drug delivery. So far, applications have relied on direct coupling with therapeutic agents. In the present study, we have investigated the functionalization of spherulites by STxB and the intracellular trafficking of these structures. Results. We demonstrate that STxB‐spherulites (ST×B‐functionalized spherulites) are internalized into HeLa cells in a receptor‐dependent manner. The intracellular distribution was studied by confocal microscopy for lipids, ligand and content. We observed an early separation between spherulites and STxB, leading to a late endosomal/lysosomal localization of lipids and content, whereas STxB remained partially at the plasma membrane. Conclusions. Although recognition of Gb3 is the cause of their specific adhesion to cell membranes, STxB‐spherulites do not follow the retrograde transport route. Our results strongly suggest that STxB‐spherulites are, at least in part, disrupted at the plasma membrane, leading to lipid and content targeting to the classical endocytic pathway. We discuss how these findings influence the development of innovative delivery strategies.     

Intracellular trafficking of TRP channels

Thirteen years ago, it was suggested that exocytotic insertion of store-operated channels into the plasma membrane lead to increased Ca(2+) entry in non-excitable cells upon G protein-coupled or tyrosine kinase receptor stimulation. Since the discovery of the TRP channel superfamily and their involvement in receptor-induced Ca(2+) entry, many studies have shown that different members of the TRP superfamily translocate into the plasma membrane upon stimulation. While the exact molecular mechanism by which TRP channels insert into the plasma membrane is unknown, TRP-binding proteins have been shown to directly regulate this trafficking. This review summarizes recent advances related to the mechanism of TRP channel trafficking, focusing on the role of TRP-binding proteins.