A cargo sorting receptor mediates chloroplast protein trafficking through the secretory pathway.

Nucleus-encoded chloroplast proteins can be transported via the secretory pathway. The molecular mechanisms underlying the trafficking of chloroplast proteins between the intracellular compartments are largely unclear, and a cargo sorting receptor has not previously been identified in the secretory pathway. Here we report a cargo sorting receptor that is specifically present in Viridiplantae and mediates the transport of cargo proteins to the chloroplast. Using a forward genetic analysis, we identified a gene encoding a transmembrane protein (MtTP930) in barrel medic (Medicago truncatula). Mutation of MtTP930 resulted in impaired chloroplast function and a dwarf phenotype. MtTP930 is highly expressed in the aerial parts of the plant and is localized to the ER exit sites (ERESs) and Golgi. MtTP930 contains typical cargo sorting receptor motifs, interacts with Sar1, Sec12 and Sec24, and participates in coat protein II (COPII) vesicular transport. Importantly, MtTP930 can recognize the cargo proteins plastidial N-glycosylated nucleotide pyrophosphatase/ phosphodiesterase (MtNPP) and α-carbonic anhydrase (MtCAH) in the ER, and then transport them to the chloroplast via the secretory pathway. Mutation of a homolog of MtTP930 in Arabidopsis (Arabidopsis thaliana) resulted in a similar dwarf phenotype. Furthermore, MtNPP-GFP failed to localize to chloroplasts when transgenically expressed in Attp930 protoplasts, implying that these cargo sorting receptors are conserved in plants. These findings fill a gap in our understanding of the mechanism by which chloroplast proteins are sorted and transported via the secretory pathway.

The Endosomal Sorting Complex, ESCRT, has diverse roles in blood progenitor maintenance, lineage choice and immune response.

Most hematological malignancies are associated with reduced expression of one or more components of the Endosomal Sorting Complex Required for Transport (ESCRT). However, the roles of ESCRT in stem cell and progenitor maintenance are not resolved. Parsing signaling pathways in relation to the canonical role of ESCRT poses a challenge. The Drosophila hematopoietic organ, the larval lymph gland, provides a path to dissect the roles of cellular trafficking pathways such as ESCRT in blood development and maintenance. Drosophila has 13 core ESCRT components. Knockdown of individual ESCRTs showed that only Vps28 and Vp36 were required in all lymph gland progenitors. Using the well-conserved ESCRT-II complex as an example of the range of phenotypes seen upon ESCRT depletion, we show that ESCRTs have cell-autonomous as well as non-autonomous roles in progenitor maintenance and differentiation. ESCRT depletion also sensitized posterior lobe progenitors to respond to immunogenic wasp infestation. We also identify key heterotypic roles for ESCRT in position-dependent control of Notch activation to suppress crystal cell differentiation. Our study shows that the cargo sorting machinery determines the identity of progenitors and their adaptability to the dynamic microenvironment. These mechanisms for control of cell fate may tailor developmental diversity in multiple contexts.

High-throughput analysis of glycan sorting into extracellular vesicles.

Extracellular vesicles (EVs) are cell-released vesicles that mediate intercellular communication by transferring bioactive cargo. Protein and RNA sorting into EVs has been extensively assessed, while selective enrichment of glycans in EVs remains less explored. In this study, a mass spectrometry-based approach, glycan node analysis (GNA), was applied to broadly assess the sorting of glycan features into EVs. Two metastatic variants (lung and bone) generated in mouse modes from the MDA-MB-231 human breast cancer cell line were assessed, as these EVs are known to contain distinct organotropic biomolecules. EVs were isolated from conditioned cell culture medium by tangential flow filtration and authenticated by standard techniques. GNA analysis revealed selective enrichment of several glycan features in EVs compared to the originating cells, particularly those associated with binding to the extracellular matrix, which was also observed in EVs from the parental MDA-MB-231 cell line (human pleural metastases). The bone-tropic variant displayed enrichment of distinct EV glycan features compared to the lung-tropic one. Additionally, the metastatic variants generated in mouse models displayed reduced EV glycan sorting compared to the parental metastatic cell line. This study represents the first comprehensive assessment of differences in glycan features between EVs and originating cells and provides evidence that the diversity of EV glycan sorting is reduced upon generation of variant cell lines in mouse models. Future research is likely to uncover novel mechanisms of EV glycan sorting, shed light on glycan features for EV authentication or biomarker purposes, and assess functional roles of the EV glycocode in (patho)physiology.

The ins-and-outs of exosome biogenesis, secretion, and internalization.

Exosomes are specialized cargo delivery vesicles secreted from cells by fusion of multivesicular bodies (MVBs) with the plasma membrane (PM). While the function of exosomes during physiological and pathological events has been extensively reported, there remains a lack of understanding of the mechanisms that regulate exosome biogenesis, secretion, and internalization. Recent technological and methodological advances now provide details about MVB/exosome structure as well as the pathways of exosome biogenesis, secretion, and uptake. In this review, we outline our current understanding of these processes and highlight outstanding questions following on recent discoveries in the field.

NDFIP1 limits cellular TAZ accumulation via exosomal sorting to inhibit NSCLC proliferation.

NDFIP1 has been previously reported as a tumor suppressor in multiple solid tumors, but the function of NDFIP1 in NSCLC and the underlying mechanism are still unknown. Besides, the WW domain containing proteins can be recognized by NDFIP1, resulted in the loading of the target proteins into exosomes. However, whether WW domain-containing transcription regulator 1 (WWTR1, also known as TAZ) can be packaged into exosomes by NDFIP1 and if so, whether the release of this oncogenic protein via exosomes has an effect on tumor development has not been investigated to any extent. Here, we first found that NDFIP1 was low expressed in NSCLC samples and cell lines, which is associated with shorter OS. Then, we confirmed the interaction between TAZ and NDFIP1, and the existence of TAZ in exosomes, which requires NDFIP1. Critically, knockout of NDFIP1 led to TAZ accumulation with no change in its mRNA level and degradation rate. And the cellular TAZ level could be altered by exosome secretion. Furthermore, NDFIP1 inhibited proliferation in vitro and in vivo, and silencing TAZ eliminated the increase of proliferation caused by NDFIP1 knockout. Moreover, TAZ was negatively correlated with NDFIP1 in subcutaneous xenograft model and clinical samples, and the serum exosomal TAZ level was lower in NSCLC patients. In summary, our data uncover a new tumor suppressor, NDFIP1 in NSCLC, and a new exosome-related regulatory mechanism of TAZ.

The clathrin adaptor complex-1 and Rab12 regulate post-golgi trafficking of WT epidermal growth factor receptor (EGFR).

The epidermal growth factor receptor (EGFR) plays important roles in cancer progression and is one of the major drug targets for targeted cancer therapy. Although fundamentally important, how newly synthesized EGFR is delivered to the cell surface to perform its cellular functions remains to be further investigated. In this study, we found using the approaches of gene knockout, siRNA knockdown, streptavidin pull-down, and co-immunoprecipitation assays that the clathrin adaptor complex-1 (AP-1) and Rab12 interact with EGFR and regulate the export of EGFR out of the trans-Golgi network (TGN). In addition, the tyrosine residue at the 998 position on human EGFR is critical to bind to AP-1, and this residue is important for TGN export of EGFR. We demonstrate that AP-1 and Rab12 are important for epidermal growth factor-induced phosphorylation of EGFR, cell elongation, and proliferation, suggesting that AP-1-mediated and Rab12-mediated post-Golgi trafficking is important for EGFR signaling. Moreover, TGN export of the constitutively activated mutant form of EGFR (EGFRL858R) is independent of AP-1 and Rab12. Our results reveal insights into the molecular mechanisms that mediate the TGN-to-cell surface delivery of EGFR and indicate that TGN export of WT EGFR and EGFRL858R depends on different cellular factors.

The Machinery of Exosomes: Biogenesis, Release, and Uptake.

Exosomes are a subtype of membrane-contained vesicles 40-200 nm in diameter that are secreted by cells into their surroundings. By transporting proteins, lipids, mRNA, miRNA, lncRNA, and DNA, exosomes are able to perform such vital functions as maintaining cellular homeostasis, removing cellular debris, and facilitating intercellular and interorgan communication. Exosomes travel in all body fluids and deliver their molecular messages in autocrine, paracrine as well as endocrine manners. In recent years, there has been an increased interest in studying exosomes as diagnostic markers and therapeutic targets, since in many disease conditions this machinery becomes dysregulated or hijacked by pathological processes. Additionally, delivery of exosomes and exosomal miRNA has already been shown to improve systemic metabolism and inhibit progression of cancer development in mice. However, the subcellular machinery of exosomes, including their biogenesis, release and uptake, remains largely unknown. This review will bring molecular details of these processes up to date with the goal of expanding the knowledge basis for designing impactful exosome experiments in the future.

The cell type dependent sorting of CD9- and CD81 to extracellular vesicles can be exploited to convey tumor sensitive cargo to target cells.

Extracellular vesicles (EVs) are lipid membrane-bound particles involved in cell-to-cell communication through a delivery of regulatory molecules essential for physiological processes. Since EVs efficiently vectorize specific cargo molecules, they have been proposed as suitable vehicles for therapeutic agents. Drug loading into EVs can be achieved by active, exogenous strategies or by genetic modifications of vesicle-producing cells. With the aim to produce EVs conveying therapeutic proteins, we genetically engineered and compared HEK293 to tumor cells. Tetraspanin-based RFP fusions were found to be more stable and preferentially sorted into EVs in HEK293. EVs isolated from genetically modified HEK293 cells media were captured by cancer cells, efficiently delivering their cargo. Cathepsin B cleavage site introduced between CD9/CD81 and RFP was recognized by tumor specific proteases allowing the release of the reporter protein. Our results indicate HEK293 cells as a preferential system for the production of EVs and pave the way to the development of nano-platforms for the efficient delivery of therapeutic proteins and prodrugs to tumor cells.

Reconstitution of Vesicle Budding from the TGN and Immunoisolation of Vesicles Enriched with a Specific Cargo Client.

The trans-Golgi network (TGN) is an important cargo sorting station in the secretory pathway. To reveal insights into protein sorting at the TGN, it is important to develop an assay to quantify the efficiency of cargo capture. Here, we describe an experimental approach to reconstitute the packaging of cargo proteins into vesicles at the TGN in vitro. We also describe an experimental approach to immunoisolate vesicles enriched with a specific transmembrane cargo client from the in vitro vesicle formation assay. These assays provide robust tools to directly measure the enrichment of cargo proteins into TGN-derived vesicles and to reveal novel factors that regulate TGN export process.

NDFIP1 limits cellular TAZ accumulation via exosomal sorting to inhibit NSCLC proliferation

NDFIP1 has been previously reported as a tumor suppressor in multiple solid tumors, but the function of NDFIP1 in NSCLC and the underlying mechanism are still unknown. Besides, the WW domain containing proteins can be recognized by NDFIP1, resulted in the loading of the target proteins into exosomes. However, whether WW domain-containing transcription regulator 1 (WWTR1, also known as TAZ) can be packaged into exosomes by NDFIP1 and if so, whether the release of this oncogenic protein via exosomes has an effect on tumor development has not been investigated to any extent. Here, we first found that NDFIP1 was low expressed in NSCLC samples and cell lines, which is associated with shorter OS. Then, we confirmed the interaction between TAZ and NDFIP1, and the existence of TAZ in exosomes, which requires NDFIP1. Critically, knockout of NDFIP1 led to TAZ accumulation with no change in its mRNA level and degradation rate. And the cellular TAZ level could be altered by exosome secretion. Furthermore, NDFIP1 inhibited proliferation in vitro and in vivo, and silencing TAZ eliminated the increase of proliferation caused by NDFIP1 knockout. Moreover, TAZ was negatively correlated with NDFIP1 in subcutaneous xenograft model and clinical samples, and the serum exosomal TAZ level was lower in NSCLC patients. In summary, our data uncover a new tumor suppressor, NDFIP1 in NSCLC, and a new exosome-related regulatory mechanism of TAZ.

The Regulation of Exosome Generation and Function in Physiological and Pathological Processes.

Exosomes, a type of extracellular vesicle with a diameter of approximately 100 nm that is secreted by all cells, regulate the phenotype and function of recipient cells by carrying molecules such as proteins, nucleic acids, and lipids and are important mediators of intercellular communication. Exosomes are involved in various physiological and pathological processes such as immunomodulation, angiogenesis, tumorigenesis, metastasis, and chemoresistance. Due to their excellent properties, exosomes have shown their potential application in the clinical diagnosis and treatment of disease. The functions of exosomes depend on their biogenesis, uptake, and composition. Thus, a deeper understanding of these processes and regulatory mechanisms can help to find new targets for disease diagnosis and therapy. Therefore, this review summarizes and integrates the recent advances in the regulatory mechanisms of the entire biological process of exosomes, starting from the formation of early-sorting endosomes (ESCs) by plasma membrane invagination to the release of exosomes by fusion of multivesicular bodies (MVBs) with the plasma membrane, as well as the regulatory process of the interactions between exosomes and recipient cells. We also describe and discuss the regulatory mechanisms of exosome production in tumor cells and the potential of exosomes used in cancer diagnosis and therapy.

Export of polybasic motif-containing secretory proteins BMP8A and SFRP1 from the endoplasmic reticulum is regulated by surfeit locus protein 4.

In the conventional secretory pathway, cargo receptors play important roles in exporting newly synthesized secretory proteins from the endoplasmic reticulum (ER). We previously showed that a cargo receptor, surfeit locus protein 4 (SURF4), promotes ER export of a soluble signaling molecule, sonic hedgehog, via recognizing the polybasic residues within its Cardin-Weintraub motif. In addition to sonic hedgehog, we found 30 more secretory proteins containing the polybasic motif (K/R)(K/R)(K/R)XX(K/R)(K/R), but whether SURF4 plays a general role in mediating ER export of these secretory proteins is unclear. Here, we analyzed the trafficking of four of these secretory proteins: desert hedgehog, Indian hedgehog, bone morphogenetic protein 8A (BMP8A), and secreted frizzled-related protein 1 (SFRP1). We found that the polybasic motifs contained in these cargo proteins are important for their ER export. Further analyses indicated that the polybasic motifs of BMP8A and SFRP1 interact with the triacidic motif on the predicted first luminal domain of SURF4. These interactions with SURF4 are essential and sufficient for the ER-to-Golgi trafficking of BMP8A and SFRP1. Moreover, we demonstrated that SURF4 localizes at a subpopulation of ER exit sites to regulate the ER export of its clients. Taken together, these results suggest that SURF4 is recruited to specific ER exit sites and plays a general role in capturing polybasic motif-containing secretory cargo proteins through electrostatic interactions.

Overlapping Machinery in Lysosome-Related Organelle Trafficking: A Lesson from Rare Multisystem Disorders.

Lysosome-related organelles (LROs) are a group of functionally diverse, cell type-specific compartments. LROs include melanosomes, alpha and dense granules, lytic granules, lamellar bodies and other compartments with distinct morphologies and functions allowing specialised and unique functions of their host cells. The formation, maturation and secretion of specific LROs are compromised in a number of hereditary rare multisystem disorders, including Hermansky-Pudlak syndromes, Griscelli syndrome and the Arthrogryposis, Renal dysfunction and Cholestasis syndrome. Each of these disorders impacts the function of several LROs, resulting in a variety of clinical features affecting systems such as immunity, neurophysiology and pigmentation. This has demonstrated the close relationship between LROs and led to the identification of conserved components required for LRO biogenesis and function. Here, we discuss aspects of this conserved machinery among LROs in relation to the heritable multisystem disorders they associate with, and present our current understanding of how dysfunctions in the proteins affected in the disease impact the formation, motility and ultimate secretion of LROs. Moreover, we have analysed the expression of the members of the CHEVI complex affected in Arthrogryposis, Renal dysfunction and Cholestasis syndrome, in different cell types, by collecting single cell RNA expression data from the human protein atlas. We propose a hypothesis describing how transcriptional regulation could constitute a mechanism that regulates the pleiotropic functions of proteins and their interacting partners in different LROs.

Molecular interplays of the Entamoeba histolytica endosomal sorting complexes required for transport during phagocytosis.

Entamoeba histolytica, the causative agent of human amoebiasis, exhibits a continuous membrane remodelling to exert its virulence properties. During this dynamic process, the Endosomal Sorting Complexes Required for Transport (ESCRT) machinery is a key player, particularly in phagocytosis, a virulence hallmark of this parasite. In addition to ESCRT, other molecules contribute to membrane remodelling, including the EhADH adhesin, EhRabs, actin, and the lysobisphosphatidic acid (LBPA). The endocytosis of a prey or molecules induces membrane invaginations, resulting in endosome and multivesicular bodies (MVBs) formation for cargo delivery into lysosomes. Alternatively, some proteins are recycled or secreted. Most of these pathways have been broadly characterized in other biological systems, but poorly described in protozoan parasites. Here, we encompass 10 years of ESCRT research in E. histolytica, highlighting the role of the ESCRT-I and ESCRT-III components and the EhADH and EhVps4-ATPase accessory proteins during phagocytosis. In particular, EhADH exhibits a multifunctional role along the endocytic pathway, from cargo recognition to endosome maturation and lysosomal degradation. Interestingly, the interaction of EhADH with EhVps32 seems to shape a concurrent route to the conventional one for MVBs biogenesis, that could optimize their formation. Furthermore, this adhesin is secreted, but its role in this event remains under study. Other components from the endosomal pathway, such as EhVps23 and LBPA, are also secreted. A proteomic approach performed here, using an anti-LBPA antibody, revealed that some proteins related to membrane trafficking, cellular transport, cytoskeleton dynamics, and transcriptional and translational functions are secreted and associated to LBPA. Altogether, the accumulated knowledge around the ESCRT machinery in E. histolytica, points it out as a dynamic platform facilitating the interaction of molecules participating in different cellular events. Seen as an integrated system, ESCRTs lead to a better understanding of E. histolytica phagocytosis.

Exosomal MicroRNAs as Novel Cell-Free Therapeutics in Tissue Engineering and Regenerative Medicine.

Extracellular vesicles (EVs) are membrane-bound vesicles (50-1000 nm) that can be secreted by all cell types. Microvesicles and exosomes are the major subsets of EVs that exhibit the cell-cell communications and pathological functions of human tissues, and their therapeutic potentials. To further understand and engineer EVs for cell-free therapy, current developments in EV biogenesis and secretion pathways are discussed to illustrate the remaining gaps in EV biology. Specifically, microRNAs (miRs), as a major EV cargo that exert promising therapeutic results, are discussed in the context of biological origins, sorting and packing, and preclinical applications in disease progression and treatments. Moreover, advanced detection and engineering strategies for exosomal miRs are also reviewed. This article provides sufficient information and knowledge for the future design of EVs with specific miRs or protein cargos in tissue repair and regeneration.

Delineating the role of extracellular vesicles in cancer metastasis: A comprehensive review.

Extracellular vesicles (EVs) are subcellular messengers that aid in the formation and spread of cancer by enabling tumor-stroma communication. EVs develop from the very porous structure of late endosomes and hold information on both the intrinsic "status" of the cell and the extracellular signals absorbed by the cells from their surroundings. These EVs contain physiologically useful components, including as nucleic acids, lipids, and proteins, which have been found to activate important signaling pathways in tumor and tumor microenvironment (TME) cells, aggravating tumor growth. We highlight critical cell biology mechanisms that link EVS formation to cargo sorting in cancer cells in this review.Sorting out the signals that control EVs creation, cargo, and delivery will aid our understanding of carcinogenesis. Furthermore, we reviewed how cancer development and spreading behaviors are affected by coordinated communication between malignant and non-malignant cells. Herein, we studied the reciprocal exchanges via EVs in various cancer types. Further research into the pathophysiological functions of various EVs in tumor growth is likely to lead to the discovery of new biomarkers in liquid biopsy and the development of tumor-specific therapies.

Current Knowledge on Exosome Biogenesis, Cargo-Sorting Mechanism and Therapeutic Implications.

Extracellular vesicles (EVs) are nanoscale membrane vesicles released by donor cells that can be taken up by recipient cells. The study of EVs has the potential to identify unknown cellular and molecular mechanisms in intercellular communication and disease. Exosomes, with an average diameter of ≈100 nanometers, are a subset of EVs. Different molecular families have been shown to be involved in the formation of exosomes and subsequent secretion of exosomes, which largely leads to the complexity of the form, structure and function of exosomes. In addition, because of their low immunogenicity and ability to transfer a variety of bioactive components to recipient cells, exosomes are regarded as effective drug delivery systems. This review summarizes the known mechanisms of exosomes biogenesis, cargo loading, exosomes release and bioengineering, which is of great importance for further exploration into the clinical applications of EVs.

A SURF4-to-proteoglycan relay mechanism that mediates the sorting and secretion of a tagged variant of sonic hedgehog.

SignificanceSonic Hedgehog (Shh) is a key signaling molecule that plays important roles in embryonic patterning, cell differentiation, and organ development. Although fundamentally important, the molecular mechanisms that regulate secretion of newly synthesized Shh are still unclear. Our study reveals a role for the cargo receptor, SURF4, in facilitating export of Shh from the endoplasmic reticulum (ER) via a ER export signal. In addition, our study provides evidence suggesting that proteoglycans promote the dissociation of SURF4 from Shh at the Golgi, suggesting a SURF4-to-proteoglycan relay mechanism. These analyses provide insight into an important question in cell biology: how do cargo receptors capture their clients in one compartment, then disengage at their destination?

ADP-Ribosylation Factor Family of Small GTP-Binding Proteins: Their Membrane Recruitment, Activation, Crosstalk and Functions.

Members of the ADP-ribosylation factor (ARF) family of guanine-nucleotide binding proteins play critical roles in various cellular processes, especially in regulating the secretory, and endocytic pathways. The fidelity of intracellular vesicular trafficking depends on proper activations and precise subcellular distributions of ARF family proteins regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Here we review recent progress in understanding the membrane recruitment, activation, crosstalk, and functions of ARF family proteins.

Neutral sphingomyelinase 2 controls exosome secretion by counteracting V-ATPase-mediated endosome acidification.

During endosome maturation, neutral sphingomyelinase 2 (nSMase2, encoded by SMPD3) is involved in budding of intraluminal vesicles (ILVs) into late endosomes or multivesicular bodies (MVBs). Fusion of these with the plasma membrane results in secretion of exosomes or small extracellular vesicles (sEVs). Here, we report that nSMase2 activity controls sEV secretion through modulation of vacuolar H+-ATPase (V-ATPase) activity. Specifically, we show that nSMase2 inhibition induces V-ATPase complex assembly that drives MVB lumen acidification and consequently reduces sEV secretion. Conversely, we further demonstrate that stimulating nSMase2 activity with the inflammatory cytokine TNFα (also known as TNF) decreases acidification and increases sEV secretion. Thus, we find that nSMase2 activity affects MVB membrane lipid composition to counteract V-ATPase-mediated endosome acidification, thereby shifting MVB fate towards sEV secretion. This article has an associated First Person interview with the first author of the paper.