Conserved Arabidopsis ECHIDNA protein mediates trans-Golgi-network trafficking and cell elongation

Multiple steps of plant growth and development rely on rapid cell elongation during which secretory and endocytic trafficking via the trans-Golgi network (TGN) plays a central role. Here, we identify the ECHIDNA (ECH) protein from Arabidopsis thaliana as a TGN-localized component crucial for TGN function. ECH partially complements loss of budding yeast TVP23 function and a Populus ECH complements the Arabidopsis ech mutant, suggesting functional conservation of the genes. Compared with wild-type, the Arabidopsis ech mutant exhibits severely perturbed cell elongation as well as defects in TGN structure and function, manifested by the reduced association between Golgi bodies and TGN as well as mislocalization of several TGN-localized proteins including vacuolar H(+)-ATPase subunit a1 (VHA-a1). Strikingly, ech is defective in secretory trafficking, whereas endocytosis appears unaffected in the mutant. Some aspects of the ech mutant phenotype can be phenocopied by treatment with a specific inhibitor of vacuolar H(+)-ATPases, concanamycin A, indicating that mislocalization of VHA-a1 may account for part of the defects in ech. Hence, ECH is an evolutionarily conserved component of the TGN with a central role in TGN structure and function.     

Intracellular trafficking of somatostatin receptors

The somatostatin receptor subtypes 1-5 (sst(1)-sst(5)) exhibit different intracellular trafficking and endosomal sorting after agonist exposure. The internalization of the somatostatin receptor subtypes sst(2), sst(3) and sst(5) occurs to a much higher extent after somatostatin exposure than of sst(1) or sst(4). After endocytosis, sst(2) and sst(5) recycle to the plasma membrane, whereas sst(3) is predominantly down-regulated. This review will focus on the molecular mechanisms of the differential intracellular trafficking of sst(2), sst(3) and sst(5), and discusses our current knowledge on somatostatin receptor interacting proteins.     

Tryptophan-independent auxin biosynthesis contributes to early embryogenesis in Arabidopsis

The phytohormone auxin regulates nearly all aspects of plant growth and development. Tremendous achievements have been made in elucidating the tryptophan (Trp)-dependent auxin biosynthetic pathway; however, the genetic evidence, key components, and functions of the Trp-independent pathway remain elusive. Here we report that the Arabidopsis indole synthase mutant is defective in the long-anticipated Trp-independent auxin biosynthetic pathway and that auxin synthesized through this spatially and temporally regulated pathway contributes significantly to the establishment of the apical–basal axis, which profoundly affects the early embryogenesis in Arabidopsis. These discoveries pave an avenue for elucidating the Trp-independent auxin biosynthetic pathway and its functions in regulating plant growth and development.The phytohormone auxin plays critical roles in almost every aspect of plant development including embryogenesis, architecture formation, and tropic growth. One of the most fascinating topics in plant biology is how auxin can have so many diverse and context-specific functions (1). Dynamic auxin gradients, which are regulated mainly by local auxin synthesis, catabolism, conjugation, and polar auxin transport, are essential for integration of various environmental and endogenous signals (2, 3). Auxin perception and signaling systems are responsible for a read-out of the auxin gradients (1).Local auxin biosynthesis is important for leaf development, shade avoidance, root-specific ethylene sensitivity, vascular patterning, flower patterning, and embryogenesis (4). Indole-3-acetic acid (IAA), the naturally occurring principal auxin in plants, is biosynthesized from tryptophan (Trp) through four proposed routes according to their key intermediates, namely indole-3-acetaldoxime (IAOx), indole-3-pyruvic acid (IPyA), indole-3-acetamide (IAM), and tryptamine (TAM) (5). The TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA)/YUCCA (YUC) linear pathway has been considered as a predominant Trp-dependent auxin biosynthetic pathway (4, 6–8). However, labeling studies and analyses of Trp auxotrophic mutants have long predicted the existence of a Trp-independent IAA biosynthetic pathway (9–13). When the Arabidopsis and maize seedlings were fed with isotope-labeled precursor, IAA was more enriched than Trp, and the incorporation of label into IAA from Trp is low, suggesting that IAA can be produced de novo without Trp as an intermediate (11, 12). The Trp biosynthetic mutant trp1, defective in phosphoribosylanthranilate transferase (PAT), and indole-3-glycerol phosphate synthase (IGS) antisense plants are deficient in steps earlier than indole-3-glycerol phosphate (IGP) formation and display decreased levels of both IAA and Trp (10, 14). However, the trp3 and trp2 mutants, defective in tryptophan synthase α (TSA) and tryptophan synthase β (TSB) subunits, respectively, accumulate higher levels of IAA than the wild type despite containing lower Trp levels (10, 11, 15, 16). These data strongly suggested the existence of the Trp-independent IAA biosynthetic pathway that might branch from IGP and/or indole (10). Further studies suggested that a cytosol-localized indole synthase (INS) may be involved in the Trp-independent biosynthesis of indole-containing metabolite (17). However, the molecular basis, biological functions, and spatiotemporal regulation of the Trp-independent IAA biosynthetic pathway have remained a mystery.In higher eukaryotes, embryogenesis initiates the generation of the species-specific body plan. During embryogenesis, a single-celled zygote develops into a functional multicellular organism with cells adopting specific fates according to their relative positions. In higher plants, essential architecture features, such as body axes and major tissue layers, are established in embryogenesis, and auxin plays a vital role in apical-basal patterning and embryo axis formation (18, 19). Local auxin biosynthesis, polar auxin transport facilitated by PIN-FORMED1/3/4/7 (PIN1/3/4/7), and auxin response coordinately regulate apical–basal pattern formation during embryogenesis (7, 20–22). The TAA and YUC families in Trp-dependent IAA biosynthesis predominantly regulate embryogenesis at or after the globular stage (7, 22). However, the auxin source for embryogenesis before the globular stage remains elusive. In this study, we provide, to our knowledge, the first genetic and biochemical evidence that the cytosol-localized INS is a key component in the long predicted Trp-independent auxin biosynthetic pathway and is critical for apical–basal pattern formation during early embryogenesis in Arabidopsis.     

SNX27-FERM-SNX1 complex structure rationalizes divergent trafficking pathways by SNX17 and SNX27

The molecular events that determine the recycling versus degradation fates of internalized membrane proteins remain poorly understood. Two of the three members of the SNX-FERM family, SNX17 and SNX31, utilize their FERM domain to mediate endocytic trafficking of cargo proteins harboring the NPxY/NxxY motif. In contrast, SNX27 does not recycle NPxY/NxxY-containing cargo but instead recycles cargo containing PDZ-binding motifs via its PDZ domain. The underlying mechanism governing this divergence in FERM domain binding is poorly understood. Here, we report that the FERM domain of SNX27 is functionally distinct from SNX17 and interacts with a novel DLF motif localized within the N terminus of SNX1/2 instead of the NPxY/NxxY motif in cargo proteins. The SNX27-FERM-SNX1 complex structure reveals that the DLF motif of SNX1 binds to a hydrophobic cave surrounded by positively charged residues on the surface of SNX27. The interaction between SNX27 and SNX1/2 is critical for efficient SNX27 recruitment to endosomes and endocytic recycling of multiple cargoes. Finally, we show that the interaction between SNX27 and SNX1/2 is critical for brain development in zebrafish. Altogether, our study solves a long-standing puzzle in the field and suggests that SNX27 and SNX17 mediate endocytic recycling through fundamentally distinct mechanisms.

Endosomes are key platforms for transmembrane receptor and lipid sorting as well as for cell signaling. Cell surface receptors arrive from endocytic or anterograde trafficking routes and are either sent to the lysosome for degradation or recycled to other compartments, including the plasma membrane and the trans-Golgi network (1–3). Endosomal trafficking is indispensable for maintaining plasma membrane homeostasis and is tightly regulated by many pivotal protein machineries (1–3). Consequently, dysregulation of this process contributes to the development of a variety of human diseases, including Parkinson’s disease, Alzheimer’s disease, and cancer (4–6). Finally, many intracellular pathogens, including vacuolar bacteria, human papilloma virus, and SARS-CoV-2, hijack the endosomal trafficking pathways for their infection and replication (7–10).Members of the SNX-FERM subfamily have emerged as key proteins essential for sequence-dependent cargo recycling. The SNX-FERM subfamily is defined by the presence of a FERM domain, which interacts with NPxY/NxxY motifs located within the cytoplasmic tails of many transmembrane proteins, in addition to the phox homology (PX) domain, which is involved in phosphatidylinositol binding (2, 11) (Fig. 1A). The SNX-FERM subfamily encompasses three members: SNX17, SNX27, and SNX31 (2, 11). Unique to this subfamily, SNX27 also harbors a PDZ domain that can engage with PDZ-binding motifs (PDZbm) at the C-terminus of transmembrane proteins. SNX17 is a well-established regulator for endosomal trafficking and mediates trafficking of many cargoes bearing the NPxY/NxxY motif together with retriever (VPS35L, VPS26C, and VPS29), the CCC (COMMD/CCDC22/CCDC93) complex, and the actin-regulatory WASH complex (12–17). Established SNX17 cargoes include P-selectin, LDL receptor–related protein 1 (LRP1), VLDLR, and α5β1 integrins (12, 18–20). SNX31 is less studied; however, recent studies indicate that SNX31 can function analogously to SNX17 and regulate turnover and recycling of β1 integrin (21). In contrast with SNX17 and SNX31, SNX27 is most known to regulate endosomal trafficking of PDZbm-containing cargoes, such as glucose transporter GLUT1 and the β2 adrenergic receptor (22–24). SNX27 regulates endosomal trafficking through engaging with retromer (VPS35, VPS26A/B, and VPS29), a SNX–Bin/Amphiphysin/Rvs (BAR) dimer (SNX1/2 in complex with SNX5/6), and the WASH complex (23, 25–31). Both SNX17 and SNX27 have been linked with pathogenesis of multiple neurological disorders, such as Alzheimer’s disease and Down’s syndrome, emphasizing the importance of SNX-FERM–mediating endocytic recycling in development and human diseases (32–34). Interestingly, it was reported that the FERM domain of SNX27 could interact with peptides containing the NPxY/NxxY motif (35, 36), yet unlike SNX17 and SNX31, SNX27 does not promote recycling of NPxY/NxxY motif-containing cargoes.Open in a separate windowFig. 1.SNX27 specifically binds to multiple motifs centered on DLF within the N termini of SNX1/2. (A) Domain architecture of SNX-BAR and SNX-FERM proteins used in this study. PX, BAR, FERM (band4.1-ezrin-radixin-moesin), and PDZ (postsynaptic density 95-discs large-zonula occludens). (B) MBP-SNX1-N, SNX1-ΔN, and MBP pull-down of purified His-tagged SNX27 FERM. Shown is a Coomassie Blue–stained sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS/PAGE) gel with purified proteins (Left) and bound samples (Right). (C) Steady-state localization of different YFP-SNX1 constructs. HeLa cells were transiently transfected with mCherry-SNX27 (red) and YFP or YFP-SNX1 (green). A schematic diagram of KPI-SNX1-N is present on the bottom. (Scale bar: 10 μm.) (D) Colocalization of mCherry-SNX27 and YFP in cells in C. Each dot represents Pearson’s correlation coefficients from one cell. Data were presented as mean ± SD, and P values were calculated using one-way ANOVA and Tukey’s multiple comparisons test. *P < 0.05; ****P < 0.0001. (E) Sequence alignment of multiple fragments from the N termini of SNX1 and SNX2 highlights a DLF motif. Red color indicates the conserved DLF or DIF residues. The NPxY/NxxY motif in P-selectin is colored in orange and used for comparison.To investigate this paradox, we utilized a holistic approach, combining biochemical, structural, and cellular studies with in vivo animal models. We show that the FERM domains of the SNX-FERM family have distinct functions: whereas the FERM domain of SNX17 recognizes the NPxY/NxxY motif, the same domain in SNX27 specifically binds to a DLF motif located within the N terminus of SNX1/2. The interaction between SNX27 and SNX1/2 is necessary for not only the recruitment of SNX27 to endosomes but also the trafficking of membrane proteins, such as GLUT1 and TRAILR1. Finally, we show that interaction between SNX27 and SNX1/2 is critical for brain development in zebrafish, and the FERM domains of SNX17 and SNX27 are not interchangeable. Together, our data demonstrate that members of the SNX-FERM family regulate distinct endosomal trafficking routes via functionally divergent FERM domains and provide a unified answer for many different observations in the field.     

Asymmetric gibberellin signaling regulates vacuolar trafficking of PIN auxin transporters during root gravitropism

Gravitropic bending of plant organs is mediated by an asymmetric signaling of the plant hormone auxin between the upper and lower side of the respective organ. Here, we show that also another plant hormone, gibberellic acid (GA), shows asymmetric action during gravitropic responses. Immunodetection using an antibody against GA and monitoring GA signaling output by downstream degradation of DELLA proteins revealed an asymmetric GA distribution and response with the maximum at the lower side of gravistimulated roots. Genetic or pharmacological manipulation of GA levels or response affects gravity-mediated auxin redistribution and root bending response. The higher GA levels at the lower side of the root correlate with increased amounts of PIN-FORMED2 (PIN2) auxin transporter at the plasma membrane. The observed increase in PIN2 stability is caused by a specific GA effect on trafficking of PIN proteins to lytic vacuoles that presumably occurs downstream of brefeldin A-sensitive endosomes. Our results suggest that asymmetric auxin distribution instructive for gravity-induced differential growth is consolidated by the asymmetric action of GA that stabilizes the PIN-dependent auxin stream along the lower side of gravistimulated roots.     

Redirecting intracellular trafficking and the secretion pattern of FSH dramatically enhances ovarian function in mice

FSH and luteinizing hormone (LH) are secreted constitutively or in pulses, respectively, from pituitary gonadotropes in many vertebrates, and regulate ovarian function. The molecular basis for this evolutionarily conserved gonadotropin-specific secretion pattern is not understood. Here, we show that the carboxyterminal heptapeptide in LH is a gonadotropin-sorting determinant in vivo that directs pulsatile secretion. FSH containing this heptapeptide enters the regulated pathway in gonadotropes of transgenic mice, and is released in response to gonadotropin-releasing hormone, similar to LH. FSH released from the LH secretory pathway rescued ovarian defects in Fshb-null mice as efficiently as constitutively secreted FSH. Interestingly, the rerouted FSH enhanced ovarian follicle survival, caused a dramatic increase in number of ovulations, and prolonged female reproductive lifespan. Furthermore, the rerouted FSH vastly improved the in vivo fertilization competency of eggs, their subsequent development in vitro and when transplanted, the ability to produce offspring. Our study demonstrates the feasibility to fine-tune the target tissue responses by modifying the intracellular trafficking and secretory fate of a pituitary trophic hormone. The approach to interconvert the secretory fate of proteins in vivo has pathophysiological significance, and could explain the etiology of several hormone hyperstimulation and resistance syndromes.During vertebrate evolution, the female reproductive pattern underwent a remarkable transition from spawning of large number of eggs in primitive species under favorable conditions to more tightly controlled ovarian cycles in higher vertebrates, such that only a limited number (rodents) or a single (human and nonhuman primates) egg is released per cycle (1, 2). Coincident with this event, the single pituitary gonadotropic hormone that exists in primitive vertebrates has given rise to two gonadotropins, FSH and luteinizing hormone (LH), which coordinate gametogenesis and steroidogenesis (3–7). FSH and LH are heterodimeric glycoproteins that contain a common α-subunit and a hormone-specific β-subunit (3). Although synthesized in the same cell, the gonadotrope, FSH is mostly constitutively released in many species, whereas LH is stored in dense core granules (DCGs) and secreted in pulses via the regulated pathway in response to gonadotropin-releasing hormone (GnRH) (8, 9). Although this pattern is evolutionarily conserved, how distinct modes of gonadotropin secretion affect ovarian development and target cell responses remain unclear. Although models in which variations in secretion of gonadotropins have been achieved in vivo (10) and in vitro, including basolateral and apically polarized secretion (11), the in vivo consequences of altered mode of gonadotropin trafficking and release (constitutive vs. regulated) from pituitary are untested. We sought to identify why the two gonadotropins, LH and FSH, expressed in the same pituitary cell have evolved to exit via different routes to regulate ovarian physiology.     

An in vitro vesicle formation assay reveals cargo clients and factors that mediate vesicular trafficking

The fidelity of protein transport in the secretory pathway relies on the accurate sorting of proteins to their correct destinations. To deepen our understanding of the underlying molecular mechanisms, it is important to develop a robust approach to systematically reveal cargo proteins that depend on specific sorting machinery to be enriched into transport vesicles. Here, we used an in vitro assay that reconstitutes packaging of human cargo proteins into vesicles to quantify cargo capture. Quantitative mass spectrometry (MS) analyses of the isolated vesicles revealed cytosolic proteins that are associated with vesicle membranes in a GTP-dependent manner. We found that two of them, FAM84B (also known as LRAT domain containing 2 or LRATD2) and PRRC1, contain proline-rich domains and regulate anterograde trafficking. Further analyses revealed that PRRC1 is recruited to endoplasmic reticulum (ER) exit sites, interacts with the inner COPII coat, and its absence increases membrane association of COPII. In addition, we uncovered cargo proteins that depend on GTP hydrolysis to be captured into vesicles. Comparing control cells with cells depleted of the cargo receptors, SURF4 or ERGIC53, we revealed specific clients of each of these two export adaptors. Our results indicate that the vesicle formation assay in combination with quantitative MS analysis is a robust and powerful tool to uncover novel factors that mediate vesicular trafficking and to uncover cargo clients of specific cellular factors.

The eukaryotic secretory pathway plays important roles in delivering a variety of newly synthesized proteins to their specific resident compartments. The fidelity of protein transport in the secretory pathway depends on accurate sorting of specific cargo proteins into transport vesicles. Defects in cargo sorting cause protein mistargeting and induce defects in establishing cell polarity, immunity, as well as other physiological processes (1).A variety of cytosolic proteins are recruited to the membrane and play important roles in the protein sorting process. These cytosolic proteins include small GTPases of the Arf family and cargo adaptors (1, 2). The Arf family GTPases cycle between a GDP-bound cytosolic state and a GTP-bound state. Upon GTP binding, Arf proteins undergo conformational changes in which the N-terminal amphipathic helix is exposed to bind membranes and the switch domains change their conformation to recruit various cytosolic cargo adaptors. Once recruited onto the membranes, these cargo adaptors recognize sorting motifs on the cargo proteins. This recognition step is important for efficiently capturing cargo proteins into vesicles.The Arf family protein, Sar1, regulates packaging of cargo proteins into vesicles at the endoplasmic reticulum (ER). GTP-bound Sar1 mediates membrane recruitment of the coat protein complex II (COPII) to capture cargo proteins (2). Soluble cargo proteins in the lumen of the ER cannot be directly recognized by COPII coat and such proteins are thought to be linked to the cargo sorting machinery on the cytosolic side by transmembrane cargo receptors. One cargo receptor in mammalian cells, ERGIC53, is a mannose lectin and functions in capturing specific N-linked glycoproteins in the lumen of the ER (3). ERGIC53 regulates ER export of blood coagulation factors V and VIII, a cathepsin-Z–related protein, and alpha1-antittrypsin (4–7). Another cargo receptor, SURF4, binds amino-terminal tripeptide motifs of soluble cargo proteins and regulates ER export of soluble cargo proteins, including the yolk protein VIT-2 in Caenorhabditis elegans (8), and PCSK9 and apolipoprotein B in mammalian cells (9–11).Although significant progress has been made in understanding the general steps of cargo sorting, the spectrum of cargo clients of a specific Arf family member, cargo adaptor, or cargo receptor remains largely underinvestigated. To deepen our understanding of protein sorting in the secretory pathway, it is important to develop a robust approach to systematically reveal cargo proteins that depend on a specific factor to be efficiently packaged into vesicles. Revealing this will provide significant insight into the functions and the specificity of cargo sorting. Since distinct cytosolic proteins are recruited to membranes by different GTP-bound Arf family proteins, systematic approaches are needed to characterize budding events associated with a specific GTP-bound Arf family protein.A cellular imaging approach, pairing analysis of cargo receptors (PAIRS), has been utilized to identify the spectrum of cargo proteins that depend on a specific cargo receptor for ER export in yeast. This analysis focused on around 150 cargo molecules labeled with fluorescent tags (12). An in vitro assay that reconstitutes packaging of cargo proteins into vesicles has been used to reveal protein profiles of vesicles budded with purified COPII or COPI proteins (13). However, this analysis did not identify any non-ER resident transmembrane proteins or secretory proteins (13). This is possibly due to an unappreciated requirement for other cytosolic factors in addition to the COP coats. Affinity chromatography has been utilized to reveal cytosolic proteins that specifically interact with GTP-bound Arf or Rab proteins (14–16). In this approach, the membranes are disrupted, which might preclude identification of membrane-associated effectors. Thus, it is important to develop additional approaches to reveal novel cytosolic proteins that associate with GTP-bound Arf proteins on membranes.Here, we used an in vitro assay to reconstitute packaging of cargo proteins into transport vesicles utilizing rat liver cytosol (RLC) as a source of cytosolic proteins. Analysis of vesicle fractions by quantitative mass spectrometry (MS) revealed cytosolic proteins that are associated with vesicles dependent on GTP or GTP-bound Sar1A, and that regulate protein trafficking. One of the identified proteins, PRRC1, regulates membrane association of the COPII coat and facilitates ER-to-Golgi trafficking. We also revealed cargo proteins that depend on specific cargo receptors, ERGIC53 or SURF4, to be efficiently packaged into vesicles. Our study indicates that the vesicle formation assay is a robust tool to reveal functional roles of specific factors in protein sorting, and to uncover novel factors that regulate vesicular trafficking in the secretory pathway.     

Bone marrow cell trafficking following intravenous administration

To address trafficking of transplanted marrow cells immediately after intravenous infusion, we examined the early fate of infused non-adherent, low-density donor bone marrow cells in a syngeneic mouse model. The presence of infused donor cells, marked with indium-111 oxine (111In), with the fluorescent dye PKH26, or by a detectable transgene marker, was evaluated at 3-48 h in a variety of tissues, including peripheral blood. All three cell-marking methods indicated a rapid (< 4 h) influx of cells into the bone marrow, liver, spleen, muscle and other tissues. Moreover, these tissues remained positive for the 48 h observation period. Interestingly, analysis of PKH26-positive cells in non-myeloablated animals demonstrated that approximately 17% of infused donor marrow cells localized to the marrow space within 15 h, whereas a smaller proportion of donor cells (approximately 1-2%) localized to the marrow in recipients preconditioned by irradiation. In an effort to enrich for cells that specifically home to the bone marrow, PKH26-labelled donor marrow cells were recovered from the first host and infused into a secondary recipient. Although this was a phenotypically undefined population of cells, no increase was observed in the relative fraction of PKH26-labelled cells returning or 'homing' to the marrow of the second recipient. Taken together, these data suggest both that marrow engraftment may be mediated by non-specific 'seeding' rather than a specific homing signal, and that efficient targeting of transplanted cells to the marrow is a complex multifaceted process.     

Innate immune control of pulmonary dendritic cell trafficking

Dendritic cells (DC) are potent antigen-presenting cells that are essential for initiating adaptive immune responses. Residing within the airway mucosa, pulmonary DC continually sample the antigenic content of inhaled air and migrate to draining lymph nodes, where they present these antigens to naive T cells. The migratory patterns of pulmonary DC are highly dependent upon inflammatory conditions in the lung. Under steady-state, or non-inflammatory, conditions, pulmonary DC undergo slow but constitutive migration to draining lymph nodes, where they remain for several days and confer antigen-specific tolerance. With the onset of pulmonary inflammation, airway DC trafficking increases dramatically, and these cells rapidly accumulate within draining lymph nodes. However, within a few days, the number of airway-derived DC in lymph nodes stabilizes or declines, even in the face of ongoing pulmonary inflammation. Here, we summarize current understanding of the molecular and cellular mechanisms underlying pulmonary DC trafficking to the lymph node and the recruitment of DC precurors to the lung. It is hoped that an improved understanding of these mechanisms will lead to novel DC-mediated therapeutic strategies to treat immune-related pulmonary disease.     

Rapid formin-mediated actin-filament elongation is essential for polarized plant cell growth

Formins are present in all eukaryotes and are essential for the creation of actin-based structures responsible for diverse cellular processes. Because multicellular organisms contain large formin gene families, establishing the physiological functions of formin isoforms has been difficult. Using RNAi, we analyzed the function of all 9 formin genes within the moss Physcomitrella patens. We show that plants lacking class II formins (For2) are severely stunted and composed of spherical cells with disrupted actin organization. In contrast, silencing of all other formins results in normal elongated cell morphology and actin organization. Consistent with a role in polarized growth, For2 are apically localized in growing cells. We show that an N-terminal phosphatase tensin (PTEN)-like domain mediates apical localization. The PTEN-like domain is followed by a conserved formin homology (FH)1-FH2 domain, known to promote actin polymerization. To determine whether apical localization of any FH1-FH2 domain mediates polarized growth, we performed domain swapping. We found that only the class II FH1-FH2, in combination with the PTEN-like domain, rescues polarized growth, because it cannot be replaced with a similar domain from a For1. We used in vitro polymerization assays to dissect the functional differences between these FH1-FH2 domains. We found that both the FH1 and the FH2 domains from For2 are required to mediate exceptionally rapid rates of actin filament elongation, much faster than any other known formin. Thus, our data demonstrate that rapid rates of actin elongation are critical for driving the formation of apical filamentous actin necessary for polarized growth.     

Coordination of plant cell division and expansion in a simple morphogenetic system

Morphogenesis in plants arises from the interplay of genetic and physical interactions within a growing network of cells. The physical aspects of cell proliferation and differentiation are genetically regulated, but constrained by mechanical interactions between the cells. Higher plant tissues consist of an elaborate three-dimensional matrix of active cytoplasm and extracellular matrix, where it is difficult to obtain direct measurements of geometry or cell interactions. To properly understand the workings of plant morphogenesis, it is necessary to have biological systems that allow simple and direct observation of these processes. We have adopted a highly simplified plant system to investigate how cell proliferation and expansion is coordinated during morphogenesis. Coleocheate scutata is a microscopic fresh-water green alga with simple anatomical features that allow for accurate quantification of morphogenetic processes. Image analysis techniques were used to extract precise models for cell geometry and physical parameters for growth. This allowed construction of a deformable finite element model for growth of the whole organism, which incorporated cell biophysical properties, viscous expansion of cell walls, and rules for regulation of cell behavior. The study showed that a simple set of autonomous, cell-based rules are sufficient to account for the morphological and dynamic properties of Coleochaete growth. A variety of morphogenetic behavior emerged from the application of these local rules. Cell shape sensing is sufficient to explain the patterns of cell division during growth. This simplifying principle is likely to have application in modeling and design for engineering of higher plant tissues.     

Detection of disseminated tumor cells in bone marrow of gastric cancer using magnetic activated cell sorting and fluorescent activated cell sorting

Background and Aim: Magnetic activated cell sorting (MACS) and fluorescent activated cell sorting (FACS) were employed to enrich and detect the gastric cancer cells from a cell line in a model system, and to enrich and detect disseminated tumor cells (DTCs) from bone marrow (BM) of patients with gastric cancer. Methods: Fifteen patients with benign gastric lesions and 35 patients with gastric cancer who received curative operations between December 2002 and June 2003 were selected. Mononuclear cells were separated from their BM. Cells from cell line OCUM‐2M were seeded with 10‐grade ratio into mononuclear cells from patients with benign gastric lesion. After labeling by MACS minibeads conjugated with cytokeratin (CK) 7/8 antibodies, anti‐CK‐fluorescein isothiocyanate (FITC), and anti‐CD45‐perdinin chlorophyll protein (PerCP), the samples were enriched twice using an MS+/RS+ positive separation column. The FACS analysis was conducted on these samples before and after MACS enrichment. The results were analyzed using clinopathological parameters. Results: Disseminated tumor cells were detected in the BM of 25 (71.43%) patients with gastric cancer. The frequencies of DTCs were 1.38 × 10^?8–2.40 × 10^?5, 2.19 × 10^?7–3.70 × 10^?5, 4.01 × 10^?6–8.57 × 10^?5 in patients with well, moderately, and poorly differentiated carcinoma, respectively (P = 0.026). Disseminated tumor cells in BM had close correlation with tumor tumor‐node‐metastasis (TNM) stage (P = 0.034) and cancer‐free survival (P = 0.035). Conclusion: Disseminated tumor cells are very common in the BM of gastric cancer patients. Poor histological differentiation and more advanced TNM stage have more DTCs in the BM of gastric cancer patients. Patients with DTCs tend to have a poor prognosis.     

PTEN regulates natural killer cell trafficking in vivo

Phosphatase and tensin homolog (PTEN) is a critical negative regulator of the phosphoinositide-3 kinase pathway, members of which play integral roles in natural killer (NK) cell development and function. However, the functions of PTEN in NK cell biology remain unknown. Here, we used an NK cell-specific PTEN-deletion mouse model to define the ramifications of intrinsic NK cell PTEN loss in vivo. In these mice, there was a significant defect in NK cell numbers in the bone marrow and peripheral organs despite increased proliferation and intact peripheral NK cell maturation. Unexpectedly, we observed a significant expansion of peripheral blood NK cells and the premature egress of NK cells from the bone marrow. The altered trafficking of NK cells from peripheral organs into the blood was due to selective hyperresponsiveness to the blood localizing chemokine S1P. To address the importance of this trafficking defect to NK cell immune responses, we investigated the ability of PTEN-deficient NK cells to traffic to a site of tumor challenge. PTEN-deficient NK cells were defective at migrating to distal tumor sites but were more effective at clearing tumors actively introduced into the peripheral blood. Collectively, these data identify PTEN as an essential regulator of NK cell localization in vivo during both homeostasis and malignancy.Natural killer (NK) cells are innate lymphoid cells critical for host defense against pathogens and antitumor responses (1–3). In the naïve mouse, NK cells are distributed among a number of hematopoietic and nonhematopoietic organs, including major reservoirs within the spleen, blood, and bone marrow (BM). Factors that orchestrate NK cell trafficking during homeostasis, including chemokine receptors and adhesion molecules, remain largely unknown. The majority of studies have focused on receptors controlling NK cell migration out of the BM, such as CXCR4 and S1P₅ (4–6). During inflammatory states, other receptors have defined roles for specific tissue homing, including CCR1, CCR5, CXCR3, CX3CR1, and CXCR6 (7). Integrin molecules, such as very late antigen-4 (VLA-4), also have specific functions in retaining NK cells within BM sinusoids (8). However, the downstream intracellular signaling pathways important for trafficking remain unclear, especially in light of the complex interplay of multiple chemotactic signals during an immune response.One family of enzymes regulating a number of chemokine receptors includes the phosphoinositide 3-kinase (PI3K) signaling pathway, which plays a broad role in regulating cellular proliferation, gene expression, survival, cytoskeleton rearrangement, and migration (9). In immune cells, the PI3K pathway may be activated downstream of a number of receptors, including cytokine receptors and G protein-coupled receptors (GPCRs), the latter of which include most chemokine receptors. Stimulation of the PI3Ks results in the generation of phosphatidylinositol phosphate lipids, such as PI(3,4,5)P₃, and subsequent recruitment and activation of downstream signaling proteins, including Vav, Akt, and PDK1 (9, 10). Exogenous inhibition of PI3Ks suppresses perforin and granzyme B polarization and NK cell cytotoxicity (11). Additionally, deficiency of the leukocyte-selective PI3K p110γ or p110δ subunit results in defective NK cell development and maturation and alters the production of IFN gamma (IFN-γ) and cytotoxicity (12–14). New evidence has also linked IL-15 to the mammalian target of rapamycin (mTOR) pathway via PI3K activation (15, 16). Thus, PI3K signaling is a critical pathway for NK cell biological processes.Two primary phosphatases oppose PI3K generation of the active secondary messenger PI(3,4,5)P₃: SH2-containing inositol-5′-phosphatase 1 (SHIP1) and phosphatase and tensin homolog (PTEN). SHIP1 specifically dephosphorylates PI(3,4,5)P₃ to PI(3,4)P₂, whereas PTEN dephosphorylates the 3′ inositol phosphate on PI(3,4,5)P₃, PI(3,4)P₂, and PI(3)P, thereby also counteracting other classes of PI3Ks (10). SHIP1^−/− BM-chimeric mice have no overt alterations in NK cell distribution, and its intrinsic role in NK cell development only affects the terminal differentiation of mature NK cells (17). However, there are no reported studies of PTEN function in NK cells to date. The effects of lymphocyte-selective PTEN deficiency in T and B cells result from increased PI3K signaling and include increased cell survival and proliferation, lowered activation threshold through the B-cell receptor (18), and loss of costimulatory requirements in T cells (19). The role of phosphatases, particularly SHIP1 and PTEN, in cellular migration, however, remains controversial and appears to be dependent on both the cell studied and the mode of stimulation (20–22). Furthermore, unique aspects of PTEN function have been reported, including protein phosphatase activity (23) and regulation via intracellular sequestration to the cytoplasmic membrane (24). As PTEN is a major nonredundant regulator of PI3K signaling, we hypothesized that disruption of PI3K inhibition would uniquely impact NK cell developmental and functional pathways.In this study, we generated NK cell-specific PTEN-deficient mice, which have diminished opposing lipid phosphatase activity to all known PI3K family members. PTEN-deficient mice display significant defects in peripheral organ and BM NK cell compartments, with a large proportion of NK cells being inappropriately localized to the blood. These effects were due in part to alterations in NK cell trafficking in vivo, a defect that also prevented their recruitment to a localized tumor challenge. These results identify a previously unidentified role for PTEN in regulating NK cell tissue distribution during both homeostasis and malignancy in vivo.     

Multitarget magnetic activated cell sorter

Magnetic selection allows high-throughput sorting of target cells based on surface markers, and it is extensively used in biotechnology for a wide range of applications from in vitro diagnostics to cell-based therapies. However, existing methods can only perform separation based on a single parameter (i.e., the presence or absence of magnetization), and therefore, the simultaneous sorting of multiple targets at high levels of purity, recovery, and throughput remains a challenge. In this work, we present an alternative system, the multitarget magnetic activated cell sorter (MT-MACS), which makes use of microfluidics technology to achieve simultaneous spatially-addressable sorting of multiple target cell types in a continuous-flow manner. We used the MT-MACS device to purify 2 types of target cells, which had been labeled via target-specific affinity reagents with 2 different magnetic tags with distinct saturation magnetization and size. The device was engineered so that the combined effects of the hydrodynamic force produced from the laminar flow and the magnetophoretic force produced from patterned ferromagnetic structures within the microchannel result in the selective purification of the differentially labeled target cells into multiple independent outlets. We demonstrate here the capability to simultaneously sort multiple magnetic tags with >90% purity and >5,000-fold enrichment and multiple bacterial cell types with >90% purity and >500-fold enrichment at a throughput of 10⁹ cells per hour.     

Rod-like bacterial shape is maintained by feedback between cell curvature and cytoskeletal localization

Cells typically maintain characteristic shapes, but the mechanisms of self-organization for robust morphological maintenance remain unclear in most systems. Precise regulation of rod-like shape in Escherichia coli cells requires the MreB actin-like cytoskeleton, but the mechanism by which MreB maintains rod-like shape is unknown. Here, we use time-lapse and 3D imaging coupled with computational analysis to map the growth, geometry, and cytoskeletal organization of single bacterial cells at subcellular resolution. Our results demonstrate that feedback between cell geometry and MreB localization maintains rod-like cell shape by targeting cell wall growth to regions of negative cell wall curvature. Pulse-chase labeling indicates that growth is heterogeneous and correlates spatially and temporally with MreB localization, whereas MreB inhibition results in more homogeneous growth, including growth in polar regions previously thought to be inert. Biophysical simulations establish that curvature feedback on the localization of cell wall growth is an effective mechanism for cell straightening and suggest that surface deformations caused by cell wall insertion could direct circumferential motion of MreB. Our work shows that MreB orchestrates persistent, heterogeneous growth at the subcellular scale, enabling robust, uniform growth at the cellular scale without requiring global organization.How cells maintain stable and defined morphologies is a fundamental question in all branches of life. Building cellular-scale structures with the correct spatial architecture and mechanical properties requires that nanometer-scale proteins have the ability to detect and alter cell shape across multiple length scales. In walled organisms such as plants (1–5), fungi (6), and bacteria (7–10), morphogenesis is often achieved through an interplay between the cytoskeleton and cell wall synthesis. A central challenge in bacterial physiology is to understand the feedback between cell shape and the coordination of wall growth by the cytoskeleton.The cell wall plays a critical mechanical role in balancing turgor stress in virtually all bacteria and is both necessary and sufficient to define cell shape (11). The bacterial cell wall is a mesh-like network of sugar strands cross-linked by peptides (11, 12). In rod-shaped Escherichia coli cells, cell wall growth occurs along the cylindrical body. Biophysical modeling has suggested that a random pattern of insertion cannot preserve cell shape (13), indicating that spatial coordination of the growth machinery is necessary for cell shape maintenance. Several lines of evidence demonstrate that the actin homolog MreB (14, 15) plays a major role in this coordination in most rod-shaped bacteria. The small molecule A22 depolymerizes MreB and causes a gradual transition from a rod-like to a spherical shape (15–17). This observation suggests that the disruption of MreB changes the patterning of new material insertion, although the nature of this change remains unknown (13). In both E. coli (10) and Bacillus subtilis (8, 9), MreB rotates around the long axis of the cell in a manner dependent on cell wall synthesis, suggesting a connection between MreB and growth. In E. coli, depletion of cell wall precursors halted MreB motion (10), and in B. subtilis, the motion of cell wall synthesis enzymes was similar to that of MreB (8, 9).However, no direct evidence has been presented to show that spatiotemporal patterns of MreB colocalize with new cell wall insertion. Moreover, we currently do not know whether MreB directly coordinates the spatial pattern of cell wall insertion, what governs the localization of MreB, or how cells maintain a straight, rod-like shape. Here, we introduce imaging methodologies to discover a feedback among cell shape, MreB localization, and cell growth that actively straightens cells. We found that MreB preferentially localized to negatively curved regions of the cell outline and was depleted from regions of positive curvature. We show that cell wall growth is colocalized with MreB, which persists at a given location for several minutes, resulting in bursts of localized growth. Consistent with an active mechanism for cell straightening, cell curvature exhibits oscillatory dynamics in which the bending direction of the cell is reversed by growth. We construct growth maps of single cells that enable the quantification of bursts of growth and find that their time scales are similar to that of MreB spatial persistence. In the presence of A22, growth becomes more homogeneous, and we directly observe insertion of material at the poles, suggesting that it is curvature-dependent localization of MreB that prevents polar growth. Integrating our experimental data into a model for cell shape maintenance, we use biophysical simulations to show that a linear curvature enrichment profile biasing growth to negatively curved regions of the cell wall tends to straighten a bent cell. Moreover, deformations in cell geometry resulting from the incorporation of new glycan strands increase the processivity of insertion, suggesting that the rotational motion of MreB itself may be a result of geometric localization.     

The embryonic node behaves as an instructive stem cell niche for axial elongation

In warm-blooded vertebrate embryos (mammals and birds), the axial tissues of the body form from a growth zone at the tail end, Hensen’s node, which generates neural, mesodermal, and endodermal structures along the midline. While most cells only pass through this region, the node has been suggested to contain a small population of resident stem cells. However, it is unknown whether the rest of the node constitutes an instructive niche that specifies this self-renewal behavior. Here, we use heterotopic transplantation of groups and single cells and show that cells not destined to enter the node can become resident and self-renew. Long-term resident cells are restricted to the posterior part of the node and single-cell RNA-sequencing reveals that the majority of these resident cells preferentially express G2/M phase cell-cycle–related genes. These results provide strong evidence that the node functions as a niche to maintain self-renewal of axial progenitors.

In higher vertebrate embryos the body axis forms in head-to-tail direction from a growth zone at the tail end, which is present from gastrula stages through to the end of axis elongation, several days later. Hensen’s node is part of this growth zone. Rather than defining a distinct cell population arising very early in development, the node represents a dynamic region at the tip of the primitive streak, which appears as a morphological “node” from HH4 (1) in chick. The initial cells that make up this region are derived from two distinct cell populations, which meet at the tip of the elongating primitive streak (HH3 to 3+ in chick) (2–4). These are then joined by cells from the epiblast lateral to the anterior streak and node (5) (at stages HH3+ to HH4) and from the primitive streak immediately caudal to the node during regression (from stage HH5) (6, 7). Although ingression of cells from adjacent epiblast along most of the length of the streak continues later into development (6), this ceases at the level of the node by HH4+ (5, 8, 9). After stage 5, the node begins to regress caudally (7), while cells exit the node to lay down the midline of the developing head–tail axis, contributing to axial (notochord) and paraxial (medial somite) mesoderm, definitive endoderm, and neural midline (floorplate) tissues (Fig. 1 A–C) (5, 10–12).Open in a separate windowFig. 1.The node confers resident behavior. (A–C) Node replacement using a GFP donor showing normal node axial fates. (D and E) Epiblast lateral to the HH3+/4 node ingresses into it and gives rise to the axis and to regressing node as resident cells. (F and G) Anterior epiblast not normally fated to enter the node behaves as lateral epiblast when forced to do so. (H and I) Anterior epiblast normally gives rise to head structures. (J and K) Lateral epiblast no longer gives rise to node-derived axial structures when prevented from entering the node. (L) Quantifying tissue contribution of lateral (D, green) versus anterior (F, blue) epiblast grafts to the host. E, endoderm; F, floorplate; MS, medial-somite; N, notochord; RC, resident cell. Transverse dashed lines show levels of accompanying sections. The field of view of the wholemount images (C, E, G, I, K) is approximately 2 mm x 5 mm.Therefore, most cells pass transiently though the node, temporarily gaining a node-like gene-expression signature, which they lose upon leaving the node (5). However, transplantation of cell groups and fate-mapping experiments in chick (10, 13–15) and mouse (16–20) during early development have suggested that the node may also contain a few resident self-renewing cells that persist within the node during axial elongation, while other cells leave (Fig. 1C, “RC”). In particular, labeling of single cells in the node has provided a few examples of cells that contribute to midline structures and appear to self-renew because one or more cells remain at the site of labeling after some progeny have left (10, 17, 21, 22). At a cell-population level, grafts of groups of cells transplanted repeatedly between older and younger tailbud regions can contribute to midline structures over two or more hosts, while again some cells remain in the tailbud (14, 19). These findings have led to the idea that some cells in the node (most likely a very small subset) may have the ability to self-renew, perhaps indefinitely, thus displaying stem cell behavior.Are the self-renewing cells a special population that arose in earlier development, or might the node act as an environment (niche) (23–25) that captures a subset of the cells that enter it and instructs them to become resident and acquire self-renewal behavior and act as stem cells (26–28)? To demonstrate self-renewal and to test whether the node is an instructive stem cell niche, it is critical to test whether an individual cell can acquire this behavior when introduced to the node environment; this has not yet been attempted. Here we address this question using transplantation of groups of cells and of single cells in vivo and single-cell RNA sequencing (scRNA-seq). We find that the tip of the primitive streak is able to impart notochord and somite identity to most or all cells that enter it, while capturing a small subset to become resident and acquire self-renewal behavior. Cells from epiblast that would never have entered the node region during normal development are able to read these cues. We also define the developmental stage at which epiblast cells lose competence to respond to node signals. Long-term resident cells are preferentially located in the posterior part of the node, and display enriched expression of G2/M cell cycle markers.