The majority of new HIV infections occur in women as a result of heterosexual intercourse, overcoming multiple innate barriers to infection within the mucosa. However, the avenues through which infection is established, and the nature of bottlenecks to transmission, have been the source of considerable investigation and contention. Using a high dose of a single round non-replicating SIV-based vector containing a novel dual reporter system, we determined the sites of infection by the inoculum using the rhesus macaque vaginal transmission model. Here we show that the entire female reproductive tract (FRT), including the vagina, ecto- and endocervix, along with ovaries and local draining lymph nodes can contain transduced cells only 48 hours after inoculation. The distribution of infection shows that virions quickly disseminate after exposure and can access target cells throughout the FRT, with an apparent preference for infection in squamous vaginal and ectocervical mucosa. JRFL enveloped virions infect diverse CD4 expressing cell types, with T cells resident throughout the FRT representing the primary target. These findings establish a new perspective that the entire FRT is susceptible and virus can reach as far as the ovary and local draining lymph nodes. Based on these findings, it is essential that protective mechanisms for prevention of HIV acquisition must be present at protective levels throughout the entire FRT to provide complete protection.

SummaryCilia are specialized surface regions of eukaryotic cells that serve a variety of functions, ranging from motility to sensation and to regulation of cell growth and differentiation. The discovery that a number of human diseases, collectively known as ciliopathies, result from defective cilium function has expanded interest in these structures. Among the many properties of cilia, motility and intraflagellar transport have been most extensively studied. The latter is the process by which multiprotein complexes associate with microtubule motors to transport structural subunits along the axoneme to and from the ciliary tip. By contrast, the mechanisms by which membrane proteins and lipids are specifically targeted to the cilium are still largely unknown. In this Commentary, we review the current knowledge of protein and lipid targeting to ciliary membranes and outline important issues for future study. We also integrate this information into a proposed model of how the cell specifically targets proteins and lipids to the specialized membrane of this unique organelle.This article is part of a Minifocus on cilia and flagella. For further reading, please see related articles: 'The primary cilium at a glance' by Peter Satir et al. (J. Cell Sci. 123,(499)(500)(501)(502)(503), 'Sensory reception is an attribute of both primary cilia and motile cilia' by Robert A. Bloodgood (J. Cell Sci. 123, 505-509), 'The perennial organelle: assembly and disassembly of the primary cilium' by E. Scott Seeley and Maxence V. Nachury (J. Cell Sci. 123,(511)(512)(513)(514)(515)(516)(517)(518) and 'Flagellar and ciliary beating: the proven and the possible' by Charles B. Lindemann and Kathleen A. Lesich (J. Cell Sci. 123, 519-528). Key words: Cilia, Intraflagellar transport, Lipid rafts, Palmitoylation, TargetingJournal of Cell Science 530 roles in ciliary targeting, rather than on those molecules that are generally involved in establishment of cell polarity or in mediating vesicular transport. We draw on examples from diverse organisms and assume that key themes, if not exact molecules, are probably common to ciliary biogenesis and targeting in all eukaryotes. Axoneme assemblyNearly all ciliary axonemes contain microtubules in either the 9 + 2 arrangement, in which 9 outer doublets surround a central pair, or the 9 + 0 arrangement, in which the central pair of microtubules is absent. Motility is driven by dynein-mediated sliding between these microtubules. In either arrangement, axoneme assembly is driven by a process known as intraflagellar transport (IFT) (Kozminski et al., 1993). The ciliary axoneme and its associated cytoskeletal elements are not elongated by the addition of subunits to the base, but by extension from the growing tip (Johnson and Rosenbaum, 1992;Song and Dentler, 2001;Stephens, 2000). IFT is responsible for the delivery of structural subunits to the ciliary tip, as well as for their recycling back to the cell body (Qin et al., 2004) (Fig. 1). These processes are mediated by two distinct (Kozminski et al., 1995;...

BackgroundColonization of the female lower genital tract with Lactobacillus provides protection against STIs and adverse pregnancy outcomes. Growth of genital Lactobacillus is postulated to depend on epithelial cell-produced glycogen. However, the amount of cell-free glycogen in genital fluid available for utilization by Lactobacillus is not known.MethodsEighty-five genital fluid samples from 7 pre-menopausal women taken over 4–6 weeks were obtained using the Instead SoftCup® (EvoFem, Inc., San Diego, CA, USA) by consented donors. Cell-free glycogen and glucose in genital fluids and estrogen and progesterone in blood were quantified.FindingsGlycogen ranged from 0.1–32 μg/μl. There were significant differences between women in glycogen over the observation period. There was a strong negative correlation between glycogen and vaginal pH (r = -0.542, p<0.0001). In multivariable analysis, free glycogen levels were significantly negatively associated with both vaginal pH and progesterone (p < 0.001 and p = 0.004, respectively). Estrogen, glucose, age, sexual intercourse 24 hours prior to visit, and days after the initial visit were not significantly associated with free glycogen levels.ConclusionCell-free glycogen concentrations can be very high, up to 3% of genital fluid, and are strongly associated with acidic vaginal pH. However, the fluctuations in glycogen levels in individuals and differences between individuals do not appear to be associated with estrogen.

Eukaryotic cilia and flagella are membranous organelles that project from the cell surface and have multiple functions, including powering cell motility, the movement of extracellular fluid across the cell surface, and cell signaling (1). A number of human disorders, collectively known as the ciliopathies, have been linked to mutations in genes involved in ciliary biogenesis and function (2). Because cilia lack the machinery for protein synthesis, ciliary proteins must be synthesized outside of this organelle and then imported (3). Kinetoplastid protozoa, besides being of interest for their global health burden, serve as excellent model organisms for the elucidation of mechanisms underlying ciliary trafficking.The surface of trypanosomes contains three distinct membrane domains: the cell body (pellicular) membrane, the flagellar membrane, and the flagellar pocket membrane (4 -6). The flagellar membrane is structurally and functionally distinct from the other surface membranes (3). There is an asymmetric distribution of proteins across these membrane domains, and several proteins have been identified in kinetoplastids that are heavily enriched or restricted to the flagellar membrane (7-13). Likewise, the kinetoplast flagellar membrane has a unique lipid composition from the rest of the cell surface. The flagellar membrane is enriched in sterols (14 -16), glycolipids (17), and sphingolipids (18,19), all which are components of canonical lipid raft microdomains. Due to their composition, lipid rafts are relatively resistant to solubilization with cold non-ionic detergents such as Triton X-100 and hence are often experimentally defined as detergent-resistant membranes (DRMs). 5Many dually acylated proteins coalesce into lipid rafts, and raft disruption leads to mislocalization of DRM-associated flagellar proteins (20). Therefore, it has been proposed that protein association with lipid rafts might serve to recruit and/or retain flagellar membrane proteins (20).One dually acylated protein that is highly enriched in the flagellar membrane of the protozoan Trypanosoma cruzi is a 24-kDa flagellar calcium-binding protein (FCaBP). The N terminus of FCaBP, which is dually acylated with myristate and palmitate, localizes to the internal face of the flagellar membrane, and both modifications are necessary for this localization (12). The N terminus of FCaBP is also sufficient to direct the cytoplasmic GFP to the flagellum (12). Although dual acylation is required for localization of FCaBP, it is not clear whether it is sufficient or whether other properties of the N terminus contribute, perhaps by promoting an association with lipid rafts.Although a variety of membrane proteins with specific localization to the flagellum have been identified in protozoan parasites, the pathways of membrane targeting and sorting among pellicular and flagellar pocket and flagellar membranes are poorly understood. The studies presented herein further define the requirements for flagellar membrane targeting and lipid raft association, and elucida...

Phosphoinositide phospholipase C (PI-PLC) plays an essential role in cell signaling. A unique Trypanosoma cruzi PI-PLC (TcPI-PLC) is lipid-modified in its N terminus and localizes to the plasma membrane of amastigotes. Here, we show that TcPI-PLC is located onto the extracellular phase of the plasma membrane of amastigotes and that its N-terminal 20 amino acids are necessary and sufficient to target the fused GFP to the outer surface of the parasite. Mutagenesis of the predicted acylated residues confirmed that myristoylation of a glycine residue in the 2nd position and acyl modification of a cysteine in the 4th but not in the 8th or 15th position of the coding sequence are required for correct plasma membrane localization in T. cruzi epimastigotes or amastigotes. Interestingly, mutagenesis of the cysteine at the 8th position increased its flagellar localization. When expressed as fusion constructs with GFP, the N-terminal 6 and 10 amino acids fused to GFP are predominantly located in the cytosol and concentrated in a compartment that co-localizes with a Golgi complex marker. The N-terminal 20 amino acids of TcPI-PLC associate with lipid rafts when dually acylated. Taken together, these results indicate that N-terminal acyl modifications serve as a molecular addressing system for sending TcPI-PLC to the outer surface of the cell.