Using deep sequencing (deepCAGE), the FANTOM4 study measured the genome-wide dynamics of transcription-start-site usage in the human monocytic cell line THP-1 throughout a time course of growth arrest and differentiation. Modeling the expression dynamics in terms of predicted cis-regulatory sites, we identified the key transcription regulators, their time-dependent activities and target genes. Systematic siRNA knockdown of 52 transcription factors confirmed the roles of individual factors in the regulatory network. Our results indicate that cellular states are constrained by complex networks involving both positive and negative regulatory interactions among substantial numbers of transcription factors and that no single transcription factor is both necessary and sufficient to drive the differentiation process.

The delicate balance between endocytosis and recycling of the cell surface receptors (NMDAR and AMPAR) is essential for controlling their surface levels and degradation, and is regulated by numerous processes including lateral membrane diffusion, scaffolding protein interactions and posttranslational modifications. Generally the NMDARs undergo activity-dependent endocytosis within clathrin-coated vesicles. They then enter the endosomal system where they are either sorted into the degradative lysosomal pathway, or are replenished via endosomal recycling. Defects in endosomal trafficking therefore lead to perturbed homeostasis of NMDARs. Our recent findings provide a comprehensive understanding of how post-translational modifications of NMDAR define an extended electrostatic peptide code for cargo sorting and influence their interactions with the trafficking machinery. Currently, I am trying to understand the mechanistic basis of intracellular trafficking in NMDAR receptor homeostasis. In my talk, I will be discussing about some of our efforts in the basic studies of the structure and function of SNX27, a unique member of PX-FERM module, that control membrane trafficking. Additionally, I will highlight the novel role for phosphorylation of the NMDARs in promoting SNX27-retromer interactions, which may have significant implications for activity-dependent trafficking of NMDARs during synaptic potentiation.

Cell-penetrating peptides can translocate across the plasma membrane of living cells and thus are potentially useful agents in drug delivery applications. Disulfide-rich cyclic peptides also have promise in drug design because of their exceptional stability, but to date only one cyclic peptide has been reported to penetrate cells, the Momordica cochinchinensis trypsin inhibitor II (MCoTI-II). MCoTI-II belongs to the cyclotide family of plant-derived cyclic peptides that are characterized by a cyclic cystine knot motif. Previous studies in fixed cells showed that MCoTI-II could penetrate cells but kalata B1, a prototypic cyclotide from a separate subfamily of cyclotides, was bound to the plasma membrane and did not translocate into cells. Here, we show by live cell imaging that both MCoTI-II and kalata B1 can enter cells. Kalata B1 has the same cyclic cystine knot structural motif as MCoTI-II but differs significantly in sequence, and the mechanism by which these two peptides enter cells also differs. MCoTI-II appears to enter via macropinocytosis, presumably mediated by interaction of positively charged residues with phosphoinositides in the cell membrane, whereas kalata B1 interacts directly with the membrane by targeting phosphatidylethanolamine phospholipids, probably leading to membrane bending and vesicle formation. We also show that another plant-derived cyclic peptide, SFTI-1, can penetrate cells. SFTI-1 includes just 14 amino acids and, with the exception of its cyclic backbone, is structurally very different from the cyclotides, which are twice the size. Intriguingly, SFTI-1 does not interact with any of the phospholipids tested, and its mechanism of penetration appears to be distinct from MCoTI-II and kalata B1. The ability of diverse disulfide-rich cyclic peptides to penetrate cells enhances their potential in drug design, and we propose a new classification for them, i.e. cyclic cell-penetrating peptides.

In epithelia, junction proteins are endocytosed for modulation of cell-cell adhesion and cell polarity. In response to growth factors, the cell-cell adhesion protein E-cadherin is internalized from the cell surface with degradation or recycling as potential fates. However, the cellular machinery involved in cadherin internalization and recycling remains controversial. Here we investigated EGF-induced E-cadherin internalization. EGF stimulation of MCF-7 cells resulted in Rac1-modulated macropinocytosis of the E-cadherin-catenin complex into endosomal compartments that colocalized with EEA1 and the sorting nexin, SNX1. Depletion of cellular SNX1 levels by siRNA resulted in increased intracellular accumulation and turnover of E-cadherin internalized from the cell surface in response to EGF. Moreover, SNX1 was also required for efficient recycling of internalized E-cadherin and re-establishment of epithelial adhesion. Together, these findings demonstrate a role for SNX1 in retrieval of E-cadherin from a degradative endosomal pathway and in membrane trafficking pathways that regulate E-cadherin recycling.

We report that phosphoinositol-binding sorting nexin 5 (SNX5) associates with newly formed macropinosomes induced by EGF stimulation. We used the recruitment of GFP-SNX5 to macropinosomes to track their maturation. Initially, GFP-SNX5 is sequestered to discrete subdomains of the macropinosome; these subdomains are subsequently incorporated into highly dynamic, often branched, tubular structures. Time-lapse videomicroscopy revealed the highly dynamic extension of SNX5-labelled tubules and their departure from the macropinosome body to follow predefined paths towards the perinuclear region of the cell, before fusing with early endosomal acceptor membranes. The extension and departure of these tubular structures occurs rapidly over 5-10 minutes and is dependent upon intact microtubules. As the tubular structures depart from the macropinosome there is a reduction in the surface area and an increase in tension of the limiting membrane of the macropinosome. In addition to the recruitment of SNX5 to the macropinosome, Rab5, SNX1 and EEA1 are also recruited by newly formed macropinosomes, followed by the accumulation of Rab7. SNX5 forms heterodimers with SNX1 and this interaction is required for endosome association of SNX5. We propose that the departure of SNX5-positive tubules represents a rapid mechanism of recycling components from macropinosomes thereby promoting their maturation into Rab7-positive structures. Collectively these findings provide a detailed real-time characterisation of the maturation process of the macropinocytic endosome.