Janice A. Williams

Impaired Autophagy, Defective T Cell Homeostasis, and a Wasting Syndrome in Mice with a T Cell–Specific Deletion of Vps34

Autophagy plays a critical role in multiple aspects of the immune system, including the development and function of T lymphocytes. In mammalian cells, the class III PI3K vacuolar protein sorting (Vps)34 is thought to play a critical role in autophagy. However, recent studies have cast doubt on the role of Vps34 in autophagy, at least in certain cell types. To study the effects of Vps34 on autophagy in T lymphocytes, we generated mice that selectively lack Vps34 in the T cell lineage. Vps34 ablation in T cells caused profound defects in autophagic flux, resulting in accumulation of cellular organelles and apoptosis. These animals exhibited normal intrathymic development of conventional T cells, but they were profoundly impaired in the intrathymic development of invariant NKT cells. In peripheral organs, T cell–specific ablation of Vps34 had a profound impact on T cell homeostasis and function. Furthermore, aged animals developed an inflammatory wasting syndrome characterized by weight loss, intestinal inflammation, and anemia. Consistent with this phenotype, Vps34 was required for the peripheral maintenance and function of CD4+Foxp3+ regulatory T cells. Collectively, our study reveals a critical role for Vps34 in autophagy and for the peripheral homeostasis and function of T lymphocytes.

Rab11-FIP1C and Rab14 Direct Plasma Membrane Sorting and Particle Incorporation of the HIV-1 Envelope Glycoprotein Complex

The incorporation of the envelope glycoprotein complex (Env) onto the developing particle is a crucial step in the HIV-1 lifecycle. The long cytoplasmic tail (CT) of Env is required for the incorporation of Env onto HIV particles in T cells and macrophages. Here we identify the Rab11a-FIP1C/RCP protein as an essential cofactor for HIV-1 Env incorporation onto particles in relevant human cells. Depletion of FIP1C reduced Env incorporation in a cytoplasmic tail-dependent manner, and was rescued by replenishment of FIP1C. FIP1C was redistributed out of the endosomal recycling complex to the plasma membrane by wild type Env protein but not by CT-truncated Env. Rab14 was required for HIV-1 Env incorporation, and FIP1C mutants incapable of binding Rab14 failed to rescue Env incorporation. Expression of FIP1C and Rab14 led to an enhancement of Env incorporation, indicating that these trafficking factors are normally limiting for CT-dependent Env incorporation onto particles. These findings support a model for HIV-1 Env incorporation in which specific targeting to the particle assembly microdomain on the plasma membrane is mediated by FIP1C and Rab14.

Rab11a regulates syntaxin 3 localization and microvillus assembly in enterocytes.

Rab11a is a key component of the apical recycling endosome that aids in the trafficking of proteins to the luminal surface in polarized epithelial cells. Utilizing conditional Rab11a‐knockout specific to intestinal epithelial cells, and human colonic epithelial CaCo2‐BBE cells with stable Rab11a knockdown, we examined the molecular and pathological impact of Rab11a deficiency on the establishment of apical cell polarity and microvillus morphogenesis. We demonstrate that loss of Rab11a induced alterations in enterocyte polarity, shortened microvillar length and affected the formation of microvilli along the lateral membranes. Rab11a deficiency in enterocytes altered the apical localization of syntaxin 3. These data affirm the role of Rab11a in apical membrane trafficking and the maintenance of apical microvilli in enterocytes.

ABCG2pos lung mesenchymal stem cells are a novel pericyte subpopulation that contributes to fibrotic remodeling

Genesis of myofibroblasts is obligatory for the development of pathology in many adult lung diseases. Adult lung tissue contains a population of perivascular ABCG2(pos) mesenchymal stem cells (MSC) that are precursors of myofibroblasts and distinct from NG2 pericytes. We hypothesized that these MSC participate in deleterious remodeling associated with pulmonary fibrosis (PF) and associated hypertension (PH). To test this hypothesis, resident lung MSC were quantified in lung samples from control subjects and PF patients. ABCG2(pos) cell numbers were decreased in human PF and interstitial lung disease compared with control samples. Genetic labeling of lung MSC in mice enabled determination of terminal lineage and localization of ABCG2 cells following intratracheal administration of bleomycin to elicit fibrotic lung injury. Fourteen days following bleomycin injury enhanced green fluorescent protein (eGFP)-labeled lung MSC-derived cells were increased in number and localized to interstitial areas of fibrotic and microvessel remodeling. Finally, gene expression analysis was evaluated to define the response of MSC to bleomycin injury in vivo using ABCG2(pos) MSC isolated during the inflammatory phase postinjury and in vitro bleomycin or transforming growth factor-β1 (TGF-β1)-treated cells. MSC responded to bleomycin treatment in vivo with a profibrotic gene program that was not recapitulated in vitro with bleomycin treatment. However, TGF-β1 treatment induced the appearance of a profibrotic myofibroblast phenotype in vitro. Additionally, when exposed to the profibrotic stimulus, TGF-β1, ABCG2, and NG2 pericytes demonstrated distinct responses. Our data highlight ABCG2(pos) lung MSC as a novel cell population that contributes to detrimental myofibroblast-mediated remodeling during PF.

Treatment of Triple-Negative Breast Cancer with TORC1/2 Inhibitors Sustains a Drug-Resistant and Notch-Dependent Cancer Stem Cell Population.

Approximately 30% of triple-negative breast cancers (TNBC) harbor molecular alterations in PI3K/mTOR signaling, but therapeutic inhibition of this pathway has not been effective. We hypothesized that intrinsic resistance to TORC1/2 inhibition is driven by cancer stem cell (CSC)-like populations that could be targeted to enhance the antitumor action of these drugs. Therefore, we investigated the molecular mechanisms by which PI3K/mTOR inhibitors affect the stem-like properties of TNBC cells. Treatment of established TNBC cell lines with a PI3K/mTOR inhibitor or a TORC1/2 inhibitor increased the expression of CSC markers and mammosphere formation. A CSC-specific PCR array revealed that inhibition of TORC1/2 increased FGF1 and Notch1 expression. Notch1 activity was also induced in TNBC cells treated with TORC1/2 inhibitors and associated with increased mitochondrial metabolism and FGFR1 signaling. Notably, genetic and pharmacologic blockade of Notch1 abrogated the increase in CSC markers, mammosphere formation, and in vivo tumor-initiating capacity induced by TORC1/2 inhibition. These results suggest that targeting the FGFR-mitochondrial metabolism-Notch1 axis prevents resistance to TORC1/2 inhibitors by eradicating drug-resistant CSCs in TNBC, and may thus represent an attractive therapeutic strategy to improve drug responsiveness and efficacy.

The C-terminal domain of Drosophila βHeavy-spectrin exhibits autonomous membrane association and modulates membrane area

Current models of cell polarity invoke asymmetric cues that reorganize the secretory apparatus to induce polarized protein delivery. An important step in this process is the stabilization of the protein composition in each polarized membrane domain. The spectrin-based membrane skeleton is thought to contribute to such stabilization by increasing the half-life of many proteins at the cell surface. Genetic evidence is consistent with a negative role for Drosophila βHeavy-spectrin in endocytosis, but the inhibitory mechanism has not been elucidated. Here, we investigated the membrane binding properties of the C-terminal nonrepetitive domain of βHeavy-spectrin through its in vivo expression in transgenic flies. We found that this region is a membrane-association domain that requires a pleckstrin homology domain for full activity, and we showed for the first time that robust membrane binding by such a C-terminal domain requires additional contributions outside the pleckstrin homology. In addition, we showed that expression of the βHeavy-spectrin C-terminal domain has a potent effect on epithelial morphogenesis. This effect is associated with its ability to induce an expansion in plasma membrane surface area. The membrane expansions adopt a very specific bi-membrane structure that sequesters both the C-terminal domain and the endocytic protein dynamin. Our data provide supporting evidence for the inhibition of endocytosis by βHeavy-spectrin, and suggest that the C-terminal domain mediates this effect through interaction with the endocytic machinery. Spectrin may be an active partner in the stabilization of polarized membrane domains.

Conditionally immortalized colonic epithelial cell line from a Ptk6 null mouse that polarizes and differentiates in vitro

Background and Aims:  PTK6 is an intracellular src‐related tyrosine kinase that regulates differentiation in the intestine, where knockout animals have increased proliferative activity and growth characteristics. To explore the phenotype further we attempted to establish epithelial cell lines from the intestinal mucosa.

Loss of MYO5B in Mice Recapitulates Microvillus Inclusion Disease and Reveals an Apical Trafficking Pathway Distinct to Neonatal Duodenum

Background & Aims Inactivating mutations in myosin Vb (MYO5B) cause severe neonatal diarrhea in microvillus inclusion disease. Loss of active MYO5B causes the formation of pathognomonic inclusions and aberrations in brush-border enzymes. Methods We developed 3 mouse models of germline, constitutively intestinal targeted, and inducible intestinal targeted deletion of MYO5B. The mice were evaluated for enterocyte cellular morphology. Results Germline MYO5B knockout mice showed early diarrhea and failure to thrive with evident microvillus inclusions and loss of apical transporters in the duodenum. IgG was present within inclusions. Apical transporters were lost and inclusions were present in the duodenum, but were nearly absent in the ileum. VillinCre;MYO5BF/F mice showed similar pathology and morphologic changes in duodenal enterocytes. In contrast, when MYO5B KO was induced with tamoxifen treatment at 8 weeks of age, VillinCreERT2;MYO5BF/F mice developed severe diarrhea with loss of duodenal brush-border enzymes, but few inclusions were observed in enterocytes. However, if tamoxifen was administered to 2-day-old VillinCreERT2;MYO5BF/F mice, prominent microvillus inclusions were observed. Conclusions The microvillus inclusions that develop after MYO5B loss show the presence of an unrecognized apical membrane trafficking pathway in neonatal duodenal enterocytes. However, the diarrheal pathology after MYO5B loss is caused by deficits in transporter presentation at the apical membrane in duodenal enterocytes.

Annexin B9 binds to β(H)-spectrin and is required for multivesicular body function in Drosophila.

The role of the cytoskeleton in protein trafficking is still being defined. Here, we describe a relationship between the small Ca2+-dependent membrane-binding protein Annexin B9 (AnxB9), apical βHeavy-spectrin (βH) and the multivesicular body (MVB) in Drosophila. AnxB9 binds to a subset of βH spliceoforms, and loss of AnxB9 results in an increase in basolateral βH and its appearance on cytoplasmic vesicles that overlap with the MVB markers Hrs, Vps16 and EPS15. Similar colocalizations are seen when βH-positive endosomes are generated either by upregulation of βH in pak mutants or through the expression of the dominant-negative version of βH. In common with other mutations disrupting the MVB, we also show that there is an accumulation of ubiquitylated proteins and elevated EGFR signaling in the absence of AnxB9 or βH. Loss of AnxB9 or βH function also causes the redistribution of the DE-Cadherin (encoded by shotgun) to endosomal vesicles, suggesting a rationale for the previously documented destabilization of the zonula adherens in karst (which encodes βH) mutants. Reduction of AnxB9 results in degradation of the apical–lateral boundary and the appearance of the basolateral proteins Coracle and Dlg on internal vesicles adjacent to βH. These results indicate that AnxB9 and βH are intimately involved in endosomal trafficking to the MVB and play a role in maintaining high-fidelity segregation of the apical and lateral domains.

Dynamic Expansion of Gastric Mucosal Doublecortin-Like Kinase 1–Expressing Cells in Response to Parietal Cell Loss Is Regulated by Gastrin

Doublecortin-like kinase 1 (Dclk1) is considered a reliable marker for tuft cells in the gastrointestinal tract. We investigated the dynamic changes of tuft cells associated with mouse models of oxyntic atrophy and metaplasia in the stomach. Increases in the numbers of Dclk1-positive tuft cells were observed in several models of parietal cell loss. However, the expanded population of Dclk1-expressing cells showed a morphologically distinct structure in apical microvilli and acetylated microtubules, which was not seen in the tuft cells present in the normal gastric mucosa. These microvillar sensory cells (MVSCs) showed no evidence of proliferation. The expansion of the MVSCs induced by oxyntic atrophy was reversible after the return of parietal cells. More important, expansion of MVSCs after induced parietal cell loss was not observed in Gast(-/-) mice. Although the Dclk1-expressing cells in the normal gastric mucosa were in part derived from Lrig1-expressing stem cells, the Lrig1-lineaged cells did not produce the expanded Dclk1-expressing cells associated with oxyntic atrophy. These studies indicate that loss of parietal cells leads to the reversible emergence of a novel Dclk1-expressing sensory cell population in the gastric mucosa.