Golgi duplication in Trypanosoma brucei

Duplication of the single Golgi apparatus in the protozoan parasite Trypanosoma brucei has been followed by tagging a putative Golgi enzyme and a matrix protein with variants of GFP. Video microscopy shows that the new Golgi appears de novo, near to the old Golgi, about two hours into the cell cycle and grows over a two-hour period until it is the same size as the old Golgi. Duplication of the endoplasmic reticulum (ER) export site follows exactly the same time course. Photobleaching experiments show that the new Golgi is not the exclusive product of the new ER export site. Rather, it is supplied, at least in part, by material directly from the old Golgi. Pharmacological experiments show that the site of the new Golgi and ER export is determined by the location of the new basal body

Transcriptional programming of dendritic cells for enhanced MHC class II antigen presentation

6 1 Dendritic cells (DCs) orchestrate adaptive immune responses by efficiently processing and presenting pathogen-derived peptides in complex with major histocompatibility complex (MHC) class I or MHC class II molecules, resulting in the activation of functionally distinct antigen-specific CTL or helper T cells, respectively 1 . Targeted delivery of antigens in vivo to CD11b − or CD11b + DCs and the 'preferential' activation of cytotoxic or helper T cells, respectively, suggests a functional divergence among DC subsets for MHCI versus MHC class II antigen presentation that mirrors the dichotomy of effector T cells 2 . These functional differences seem to reflect cell-intrinsic features of DC subsets and are correlated with differences in expression of genes associated with the MHC class I and MHC class II antigen presentation pathways 2 . The regulatory determinants and molecular basis of functional specialization among DC subsets remain to be established. Investigations of functional heterogeneity among DC subsets in vivo have relied heavily on correlations between the expression of various cell surface markers and a variety of cellular and functional properties-for example, correlation of the expression of CD8α or CD103 on select DC subsets with the specialization of those populations for cross-presentation. As the expression of surface markers used to discriminate DC populations can vary according to their environmental niche or functional state, such analyses can result in misleading conclusions. In contrast, the transcriptional determinants and gene targets that program cellular fate and effector functions are robust to environmental perturbation. Therefore, elucidation of the gene-regulatory networks that underlie development and differentiation of DC subsets is essential to illuminate the unifying principles that govern shared and divergent functions of these populations 3 . The development of mouse models that allow genetic ablation of select DC subsets has enabled substantial advances in this regard. Deficiency of the transcription factor BATF3 results in the selective loss of resident and migratory CD11b − but not CD11b + DCs 4 . BATF3-deficient mice show impaired anti-viral and anti-tumor CTL responses while helper T cell-mediated antibody responses are unaffected 5 . These findings provide crucial genetic evidence for a predominant role of CD11b − DCs in CTL immunity. However, genetic analysis of CD11b + DCs and their proposed role in priming helper T cell immune responses has been lacking 3 . The closely related immune-specific transcription factors IRF4 and IRF8 are attractive candidates as key determinants of the functionally specialized states of DCs. IRF8 is required for the development of resident and migratory CD11b − DCs, whereas IRF4 is critical for the generation of their CD11b + counterparts 4,6-10 . In keeping with their subset-specific developmental functions, IRF4 and IRF8 are expressed reciprocally in CD11b + and CD11b − splenic, lung and gut DC population

Aquaporin-3 regulates endosome-to-cytosol transfer via lipid peroxidation for cross presentation.

Antigen cross presentation, whereby exogenous antigens are presented by MHC class I molecules to CD8+ T cells, is essential for generating adaptive immunity to pathogens and tumor cells. Following endocytosis, it is widely understood that protein antigens must be transferred from endosomes to the cytosol where they are subject to ubiquitination and proteasome degradation prior to being translocated into the endoplasmic reticulum (ER), or possibly endosomes, via the TAP1/TAP2 complex. Revealing how antigens egress from endocytic organelles (endosome-to-cytosol transfer, ECT), however, has proved vexing. Here, we used two independent screens to identify the hydrogen peroxide-transporting channel aquaporin-3 (AQP3) as a regulator of ECT. AQP3 overexpression increased ECT, whereas AQP3 knockout or knockdown decreased ECT. Mechanistically, AQP3 appears to be important for hydrogen peroxide entry into the endosomal lumen where it affects lipid peroxidation and subsequent antigen release. AQP3-mediated regulation of ECT was functionally significant, as AQP3 modulation had a direct impact on the efficiency of antigen cross presentation in vitro. Finally, AQP3-/- mice exhibited a reduced ability to mount an anti-viral response and cross present exogenous extended peptide. Together, these results indicate that the AQP3-mediated transport of hydrogen peroxide can regulate endosomal lipid peroxidation and suggest that compromised membrane integrity and coordinated release of endosomal cargo is a likely mechanism for ECT

FRET Reagent Reveals the Intracellular Processing of Peptide-Linked Antibody–Drug Conjugates

Despite the recent success of antibody–drug conjugates (ADCs) in cancer therapy, a detailed understanding of their entry, trafficking, and metabolism in cancer cells is limited. To gain further insight into the activation mechanism of ADCs, we incorporated fluorescence resonance energy transfer (FRET) reporter groups into the linker connecting the antibody to the drug and studied various aspects of intracellular ADC processing mechanisms. When comparing the trafficking of the antibody–FRET drug conjugates in various different model cells, we found that the cellular background plays an important role in how the antigen-mediated antibody is processed. Certain tumor cells showed limited cytosolic transport of the payload despite efficient linker cleavage. Our FRET assay provides a facile and robust assessment of intracellular ADC activation that may have significant implications for the future development of ADCs

FRET Reagent Reveals the Intracellular Processing of Peptide-Linked Antibody–Drug Conjugates

Despite the recent success of antibody–drug conjugates (ADCs) in cancer therapy, a detailed understanding of their entry, trafficking, and metabolism in cancer cells is limited. To gain further insight into the activation mechanism of ADCs, we incorporated fluorescence resonance energy transfer (FRET) reporter groups into the linker connecting the antibody to the drug and studied various aspects of intracellular ADC processing mechanisms. When comparing the trafficking of the antibody–FRET drug conjugates in various different model cells, we found that the cellular background plays an important role in how the antigen-mediated antibody is processed. Certain tumor cells showed limited cytosolic transport of the payload despite efficient linker cleavage. Our FRET assay provides a facile and robust assessment of intracellular ADC activation that may have significant implications for the future development of ADCs