Stalk Cells Research Articles

Matrix stiffness regulates Notch signaling activity in endothelial cells.

Notch signaling is critical for many developmental and disease-related processes. It is widely accepted that Notch has a mechanotransduction module that regulates receptor cleavage. However, the role of biomechanical properties of the cellular environment in Notch signaling in general is still poorly understood. During angiogenesis, differentiation of endothelial cells into tip and stalk cells is regulated by Notch signaling, and remodeling of the extracellular matrix occurs. We investigated the influence of substrate stiffness on the Notch signaling pathway in endothelial cells. Using stiffness-tuned polydimethylsiloxane (PDMS) substrates, we show that activity of the Notch signaling pathway inversely correlates with a physiologically relevant range of substrate stiffness (i.e. increased Notch signaling activity on softer substrates). Trans-endocytosis of the Notch extracellular domain, but not the overall endocytosis, is regulated by substrate stiffness, and integrin cell-matrix connections are both stiffness dependent and influenced by Notch signaling. We conclude that mechanotransduction of Notch activation is modulated by substrate stiffness, highlighting the role of substrate rigidity as an important cue for signaling. This might have implications in pathological situations associated with stiffening of the extracellular matrix, such as tumor growth.

Developmental Programmed Cell Death Involved in Ontogenesis of Dictamnus dasycarpus Capitate Glandular Hairs.

Plant glandular trichomes have received much attention due to their commercial and biological value. Recent studies have focused on the development of various glands in plants, suggesting that programmed cell death (PCD) may play an important role during the development of plant secretory structures. However, the development processes and cytological characteristics in different types of plant secretory structures differed significantly. This study aims to provide new data on the developmental PCD of the capitate glandular hairs in Dictamnus dasycarpus. Light, scanning, immunofluorescence labeling, and transmission electron microscopy were used to determine the different developmental processes of the capitate glandular hairs from a cytological perspective. Morphologically, the capitate glandular hair originates from one initial epidermal cell and differentiates into a multicellular trichome characterized by two basal cells, two lines of stalk cells, and a multicellular head. It is also histochemically detected by essential oils. TUNEL-positive reactions identified nuclei with diffused fluorescence or an irregular figure by DAPI, and Evans blue staining showed that the head and stalk cells lost their viability. Ultrastructural evidence revealed the developmental process by two possible modes of PCD. Non-autolytic PCD was characterized by buckling cell walls and degenerated nuclei, mitochondria, plastids, multivesicular body (MVB), and end-expanded endoplasmic reticulum in the condensed cytoplasm, which were mainly observed in the head cells. The MVB was detected in the degraded vacuole, a degraded nucleus with condensed chromatin and diffused membrane, and eventual loss of the vacuole membrane integrity exhibited typical evidence of vacuole-mediated autolytic PCD in the stalk cells. Furthermore, protoplasm degeneration coupled with dark oil droplets and numerous micro-dark osmiophilic substances was observed during late stages. The secretion mode of essential oils is also described in this paper.

Leaf sodium homeostasis controlled by salt gland is associated with salt tolerance in mangrove plant Avicennia marina.

Avicennia marina, a mangrove plant growing in coastal wetland habitats, is frequently affected by tidal salinity. To understand its salinity tolerance, the seedlings of A. marina were treated with 0, 200, 400 and 600mM NaCl. We found the whole-plant dry weight and photosynthetic parameters increased at 200mM NaCl but decreased over 400mM NaCl. The maximum quantum yield of primary photochemistry (Fv/Fm) significantly decreased at 600mM NaCl. Transmission electron microscopy observations showed high salinity caused the reduction in starch grain size, swelling of the thylakoids and separation of the granal stacks, and even destruction of the envelope. In addition, the dense protoplasm and abundant mitochondria in the secretory and stalk cells, and abundant plasmodesmata between salt gland cells were observed in the salt glands of the adaxial epidermis. At all salinities, Na+ content was higher in leaves than in stems and roots; however, Na+ content increased in the roots while it remained at a constant level in the leaves over 400mM NaCl treatment, due to salt secretion from the salt glands. As a result, salt crystals on the leaf adaxial surface increased with salinity. On the other hand, salt treatment increased Na+ and K+ efflux and decreased H+ efflux from the salt glands by the non-invasive micro-test technology, although Na+ efflux reached the maximum at 400mM NaCl. Further real-time quantitative PCR analysis indicated that the expression of Na+/H+ antiporter (SOS1 and NHX1), H+-ATPase (AHA1 and VHA-c1) and K+ channel (AKT1, HAK5 and GORK) were up-regulated, and only the only Na+ inward transporter (HKT1) was down-regulated in the salt glands enriched adaxial epidermis of the leaves under 400mM NaCl treatment. In conclusion, salinity below 200mM NaCl was beneficial to the growth of A. marina, and below 400mM, the salt glands could excrete Na+ effectively, thus improving its salt tolerance.

Analysis of Vascular Morphogenesis in Zebrafish.

Analysis of cardiovascular development in zebrafish embryos has become a major driver of vascular research in recent years. Imaging-based analyses have allowed the discovery or verification of morphologically distinct processes and mechanisms of, e.g., endothelial cell migration, angiogenic sprouting, tip or stalk cell behavior, and vessel anastomosis. In this chapter, we describe the techniques and tools used for confocal imaging of zebrafish endothelial development in combination with general experimental approaches for molecular dissection of involved signaling pathways.

Abstract 1628: Endothelial tip and stalk cell specification requires BMP9-induced βIV-spectrin expression during nascent vessel sprouting

Abstract 1628: Endothelial tip and stalk cell specification requires BMP9-induced βIV-spectrin expression during nascent vessel sprouting

Effect of Hypoxia on Branching Characteristics and Cell Subpopulations during Kidney Organ Culture.

During early developmental stages, embryonic kidneys are not fully vascularized and are potentially exposed to hypoxic conditions, which is known to influence cell proliferation and survival, ureteric bud branching, and vascularization of the developing kidney. To optimize the culture conditions of in vitro cultured kidneys and gain further insight into the effect of hypoxia on kidney development, we exposed mouse embryonic kidneys isolated at E11.5, E12.5, and E13.5 to hypoxic and normal culture conditions and compared ureteric bud branching patterns, the growth of the progenitor subpopulation hoxb7+, and the expression patterns of progenitor and differentiation markers. Branching patterns were quantified using whole organ confocal imaging and gradient-vector-based analysis. In our model, hypoxia causes an earlier expression of UB tip cell markers, and a delay in stalk cell marker gene expression. The metanephric mesenchyme (MM) exhibited a later expression of differentiation marker FGF8, marking a delay in nephron formation. Hypoxia further delayed the expression of stroma cell progenitor markers, a delay in cortical differentiation markers, as well as an earlier expression of medullary and ureteral differentiation markers. We conclude that standard conditions do not apply universally and that tissue engineering strategies need to optimize suitable culture conditions for each application. We also conclude that adapting culture conditions to specific aspects of organ development in tissue engineering can help to improve individual stages of tissue generation.

Retinoic acid signaling in mouse retina endothelial cells is required for early angiogenic growth

The development of the retinal vasculature is essential to maintain health of the tissue, but the developmental mechanisms are not completely understood. The aim of this study was to investigate the cell-autonomous role of retinoic acid signaling in endothelial cells during retina vascular development. Using a temporal and cell-specific mouse model to disrupt retinoic acid signaling in endothelial cells in the postnatal retina (Pdgfbicre/+dnRAR403fl/fl mutants), we discovered that angiogenesis in the retina is significantly decreased with a reduction in retina vascularization, endothelial tip cell number and filipodia, and endothelial ‘crowding’ of stalk cells. Interestingly, by P15, the vasculature can overcome the early angiogenic defect and fully vascularized the retina. At P60, the vasculature is intact with no evidence of retina cell death or altered blood retinal barrier integrity. Further, we identified that the angiogenic defect seen in mutants at P6 correlates with decreased Vegfr3 expression in endothelial cells. Collectively, our work identified a previously unappreciated function for endothelial retinoic acid signaling in early retinal angiogenesis.

Epithelial morphogenesis in the Drosophila egg chamber requires Parvin and ILK.

Integrins are the major family of transmembrane proteins that mediate cell-matrix adhesion and have a critical role in epithelial morphogenesis. Integrin function largely depends on the indirect connection of the integrin cytoplasmic tail to the actin cytoskeleton through an intracellular protein network, the integrin adhesome. What is currently unknown is the role of individual integrin adhesome components in epithelia dynamic reorganization. Drosophila egg chamber consists of the oocyte encircled by a monolayer of somatic follicle epithelial cells that undergo specific cell shape changes. Egg chamber morphogenesis depends on a developmental array of cell-cell and cell-matrix signalling events. Recent elegant work on the role of integrins in the Drosophila egg chamber has indicated their essential role in the early stages of oogenesis when the pre-follicle cells assemble into the follicle epithelium. Here, we have focused on the functional requirement of two key integrin adhesome components, Parvin and Integrin-Linked Kinase (ILK). Both proteins are expressed in the developing ovary from pupae to the adult stage and display enriched expression in terminal filament and stalk cells, while their genetic removal from early germaria results in severe disruption of the subsequent oogenesis, leading to female sterility. Combining genetic mosaic analysis of available null alleles for both Parvin and Ilk with conditional rescue utilizing the UAS/Gal4 system, we found that Parvin and ILK are required in pre-follicle cells for germline cyst encapsulation and stalk cell morphogenesis. Collectively, we have uncovered novel developmental functions for both Parvin and ILK, which closely synergize with integrins in epithelia.

Cancer immunotherapies transition endothelial cells into HEVs that generate TCF1+ T lymphocyte niches through a feed-forward loop.

The lack of Tcell infiltrates is a major obstacle to effective immunotherapy in cancer. Conversely, the formation of tumor-associated tertiary-lymphoid-like structures (TA-TLLSs), which are the local site of humoral and cellular immune responses against cancers, is associated with good prognosis, and they have recently been detected in immune checkpoint blockade (ICB)-responding patients. However, how these lymphoid aggregates develop remains poorly understood. By employing single-cell transcriptomics, endothelial fate mapping, and functional multiplex immune profiling, we demonstrate that antiangiogenic immune-modulating therapies evoke transdifferentiation of postcapillary venules into inflamed high-endothelial venules (HEVs) via lymphotoxin/lymphotoxin beta receptor (LT/LTβR) signaling. In turn, tumor HEVs boost intratumoral lymphocyte influx and foster permissive lymphocyte niches for PD1- and PD1+TCF1+ CD8 Tcell progenitors that differentiate into GrzB+PD1+ CD8T effector cells. Tumor-HEVs require continuous CD8 and NK cell-derived signals revealing that tumor HEV maintenance is actively sculpted by the adaptive immune system through a feed-forward loop.

Autophagy of the somatic stalk cells likely nurses the propagating spores of Dictyostelid social amoebas [version 2; peer review: 2 approved, 1 approved with reservations

Autophagy (self-feeding) assists survival of starving cells by partial self-digestion, while dormancy as cysts, spores or seeds enables long-term survival. Starving Dictyostelium amoebas construct multicellular fruiting bodies with spores and stalk cells, with many Dictyostelia still able to encyst individually like their single-celled ancestors. While autophagy mostly occurs in the somatic stalk cells, autophagy gene knock-outs in Dictyostelium discoideum (D. discoideum) formed no spores and lacked cAMP induction of prespore gene expression. To investigate whether autophagy also prevents encystation, we knocked-out autophagy genes atg5 and atg7 in the dictyostelid Polysphondylium pallidum, which forms both spores and cysts. We measured spore and cyst differentiation and viability in the knock-out as well as stalk and spore gene expression and its regulation by cAMP. We tested a hypothesis that spores require materials derived from autophagy in stalk cells. Sporulation requires secreted cAMP acting on receptors and intracellular cAMP acting on PKA. We compared the morphology and viability of spores developed in fruiting bodies with spores induced from single cells by stimulation with cAMP and 8Br-cAMP, a membrane-permeant PKA agonist. Loss of autophagy in P. pallidum reduced but did not prevent encystation. Stalk cells still differentiated but stalks were disorganised. However, no spores were formed at all and cAMP-induced prespore gene expression was lost. D. discoideum spores induced in vitro by cAMP and 8Br-cAMP were smaller and rounder than spores formed multicellularly and while they were not lysed by detergent they germinated not (strain Ax2) or poorly (strain NC4), unlike spores formed in fruiting bodies. The stringent requirement of sporulation on both multicellularity and autophagy, which occurs mostly in stalk cells, suggests that stalk cells nurse the spores through autophagy. This highlights autophagy as a major cause for somatic cell evolution in early multicellularity.

Biosynthesis of gold nanoparticles in the fruiting body of enoki mushrooms (Flammulina velutipes) under Pb2+ induction.

Fungi can produce many compounds, such as proteins, enzymes, amino acids, and polysaccharides, which are internalised and enriched for metals, and are widely used as reducing and stabilising agents for the biosynthesis of gold nanoparticles (Au NPs). Almost all fungal sources used in the synthesis of the Au NPs are in the form of cell filtrates or mycelial suspensions. However, the culture of cell-free fungal filtrate and mycelium is not comparable to the propagation of fungal substrates in input and operation. Here, we evaluated in vivo biosynthesis of Au NPs in enoki mushrooms (Flammulina velutipes). HAuCl4 was reduced in the fruiting body of the enoki mushrooms via induction by Pb2+ , resulting in the generation of Au NPs. We then employed UV-Vis absorption spectroscopy, Transmission Electron Microscope, and Energy Dispersive Spectrometer to characterise various shapes of the Au NPs. The elemental analysis indicated that the Au NPs were mainly concentrated in organelles of the stalk and cap cells. We also demonstrated that 0.3-0.5mM HAuCl4 was the optimal stress treatment concentration based on the changes in physiological indicators of the enoki mushrooms. This work reveals that fungi can be utilised well as nanomaterial bioreactors.

Abstract B018: Eosinophils alter metastatic spread in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDA) is the third most common cause of cancer death in the US with a 11% 5-year survival rate. A key contributor of this dismal prognosis is early and frequent metastatic spread with limited treatment options for patients. Metastasis in PDA is facilitated by a robust fibroinflammatory response in the primary tumor site, a large portion of which is composed of infiltrating immune cells, and survival during transit to establish in the secondary site. In the primary tumor reactive stroma, we found increased eosinophils in both human and mouse PDA compared to the normal pancreas. Eosinophils are an innate immune cell best known for their ability to control parasite responses and have been implicated in either pro- or anti-cancer roles in various solid tumors. We used the ΔdblGata mice, which lack eosinophils, to study the role of eosinophils during metastatic spread using a resectable model of PDA metastasis. This model of metastasis was developed to facilitate consistent seeding of lung metastasis in approximately 50 percent of mice from a transient subcutaneous tumor which undergoes subsequent surgical removal and harvest to evaluate metastasis at 4 weeks. We found that mice lacking eosinophils have increased metastasis to the lung in our resectable model of PDA metastasis but no difference in establishment of IV injected PDA tumor cells, suggesting eosinophil control at the primary tumor site. Single cell sequencing of the subcutaneous tumors finds differences in the angiogenic subtypes, tip and stalk cells, in mice lacking eosinophils. Sequencing of eosinophil populations in humans and mice find an increase in angiogenic signaling suggesting a regulatory role in tumor cell access to the circulation. We conclude that eosinophils in PDA inhibit the metastatic process through control of angiogenic signaling. Citation Format: Megan T. Hoffman, Patrick J. Lenehan, Jennifer Wang, Lestat Ali, Li Qiang, Amiko Uchida, Sara Vayrynen, Andressa Dias Costa, Michael Dougan, Max Heckler, Jonathan Nowak, Brian W. Wolpin, Stephanie K. Dougan. Eosinophils alter metastatic spread in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B018.

Biomimicking Leaf-Vein Engraved Soft and Elastic Membrane Promotes Vascular Reconstruction.

Hierarchical vasculature reconstruction is fundamental for tissue regeneration. The regeneration of functional vascular network requires a proper directional guidance, especially in case of large-size defects. To provide the "running track" for vasculature, a leaf-vein mimetic membrane using soft and elastic poly(lactide-co-propylene glycol-co-lactide) dimethacrylate is developed. Engraved with an interconnected and perfusable leaf-vein micropattern, the membrane can guide human umbilical vein endothelial cells (HUVECs) to form vasculature in vitro. In particular, the "running track" upregulates the angiogenesis-related gene expression and promotes the HUVECs to differentiate into tip cells and stalk cells via tuning vascular endothelial growth factor receptor 2 signaling transduction. As a proof of concept, its revascularization capability using a rat calvarial defect model in vivo is evaluated. The in vivo results demonstrate that the leaf-vein engraved membrane accelerates the formation and maturation of vasculature, leading to a hierarchical blood vessel network. With the superior pro-vasculature property, it is believed that the leaf-vein engraved membrane is not only an ideal candidate for the reconstruction of calvarial vasculature but also a promising solution for more complicated vasculature reconstruction, such as muscle, skin, and heart.

Adenylate cyclase A amplification and functional diversification during Polyspondylium pallidum development

BackgroundIn Dictyostelium discoideum (Ddis), adenylate cyclase A (ACA) critically generates the cAMP oscillations that coordinate aggregation and morphogenesis. Unlike group 4 species like Ddis, other groups do not use extracellular cAMP to aggregate. However, deletion of cAMP receptors (cARs) or extracellular phosphodiesterase (PdsA) in Polyspondylium pallidum (Ppal, group 2) blocks fruiting body formation, suggesting that cAMP oscillations ancestrally control post-aggregative morphogenesis. In group 2, the acaA gene underwent several duplications. We deleted the three Ppal aca genes to identify roles for either gene and tested whether Ppal shows transient cAMP-induced cAMP accumulation, which underpins oscillatory cAMP signalling.ResultsIn contrast to Ddis, pre-aggregative Ppal cells did not produce a pulse of cAMP upon stimulation with the cAR agonist 2′H-cAMP, but acquired this ability after aggregation. Deletion of Ppal aca1, aca2 and aca3 yielded different phenotypes. aca1ˉ cells showed relatively thin stalks, aca2ˉ showed delayed secondary sorogen formation and aca3ˉ formed less aggregation centers. The aca1ˉaca2ˉ and aca1ˉaca3ˉ mutants combined individual defects, while aca2ˉaca3ˉ and aca1ˉaca3ˉaca2ˉ additionally showed > 24 h delay in aggregation, with only few aggregates with fragmenting streams being formed. The fragments developed into small fruiting bodies with stalk and spore cells. Aggregation was restored in aca2ˉaca3ˉ and aca1ˉaca3ˉaca2ˉ by 2.5 mM 8Br-cAMP, a membrane-permeant activator of cAMP-dependent protein kinase (PKA). Like Ddis, Ppal sorogens also express the adenylate cyclases ACR and ACG. We found that prior to aggregation, Ddis acaˉ/ACG cells produced a pulse of cAMP upon stimulation with 2′H-cAMP, indicating that cAMP oscillations may not be dependent on ACA alone.ConclusionsThe three Ppal replicates of acaA perform different roles in stalk morphogenesis, secondary branch formation and aggregation, but act together to enable development by activating PKA. While even an aca1ˉaca3ˉaca2ˉ mutant still forms (some) fruiting bodies, suggesting little need for ACA-induced cAMP oscillations in this process, we found that ACG also mediated transient cAMP-induced cAMP accumulation. It, therefore, remains likely that post-aggregative Ppal morphogenesis is organized by cAMP oscillations, favouring a previously proposed model, where cAR-regulated cAMP hydrolysis rather than its synthesis dominates oscillatory behaviour.

Regulation of Archease by the mTOR-vATPase axis.

Larval terminal cells of the Drosophila tracheal system generate extensive branched tubes, requiring a huge increase in apical membrane. We discovered that terminal cells compromised for apical membrane expansion - mTOR-vATPase axis and apical polarity mutants - were invaded by the neighboring stalk cell. The invading cell grows and branches, replacing the original single intercellular junction between stalk and terminal cell with multiple intercellular junctions. Here, we characterize disjointed, a mutation in the same phenotypic class. We find that disjointed encodes Drosophila Archease, which is required for the RNA ligase (RtcB) function that is essential for tRNA maturation and for endoplasmic reticulum stress-regulated nonconventional splicing of Xbp1 mRNA. We show that the steady-state subcellular localization of Archease is principally nuclear and dependent upon TOR-vATPase activity. In tracheal cells mutant for Rheb or vATPase loci, Archease localization shifted dramatically from nucleus to cytoplasm. Further, we found that blocking tRNA maturation by knockdown of tRNAseZ also induced compensatory branching. Taken together, these data suggest that the TOR-vATPase axis promotes apical membrane growth in part through nuclear localization of Archease, where Archease is required for tRNA maturation.

Chemonastic Stalked Glands in the Carnivorous Rainbow Plant Byblis gigantea LINDL. (Byblidaceae, Lamiales).

Carnivorous rainbow plants (Byblis, Byblidaceae, Lamiales) possess sticky flypaper traps for the capture, retention, and digestion of prey (mainly small insects). The trapping system is based on a multitude of millimeter-sized glandular trichomes (also termed stalked glands), which produce adhesive glue drops. For over a century, the trapping system of Byblis was considered passive, meaning that no plant movement is involved. Recently, a remarkable discovery was made: the stalked glands of Byblis are indeed capable of reacting to chemical (protein) stimuli with slow movement responses. This prompted us to investigate this phenomenon further with a series of experiments on the stimulation, kinematics, actuation, and functional morphology of the stalked glands of cultivated Byblis gigantea plants. Measured stalked gland lengths and densities on the trap leaves are similar to the data from the literature. Motion reactions could only be triggered with chemical stimuli, corroborating the prior study on the stalked gland sensitivity. Reaction time (i.e., time from stimulation until the onset of motion) and movement duration are temperature-dependent, which hints towards a tight physiological control of the involved processes. The stalked gland movement, which consist of a sequence of twisting and kinking motions, is rendered possible by the components of the stalk cell wall and is furthermore anatomically and mechanically predetermined by the orientation of cellulose microfibrils in the cell wall. Successive water displacement processes from the stalk cell into the basal cells actuate the movement. The same kinematics could be observed in stalked glands drying in air or submersed in a saturated salt solution. Stimulated and dried stalked glands as well as those from the hypertonic medium were capable of regaining their initial shape by rehydration in water. However, no glue production could be observed afterwards. The long-time overlooked chemonastic movements of stalked glands may help Byblis to retain and digest its prey; however, further research is needed to shed light on the ecological characteristics of the rainbow plant’s trapping system.

Generating polyketide diversity in Dictyostelium: a Steely hybrid polyketide synthase produces alternate products at different developmental stages.

The soil is a rich ecosystem where many ecological interactions are mediated by small molecules, and in which amoebae are low-level predators and also prey. The social amoeba Dictyostelium discoideum has a high genomic potential for producing polyketides to mediate its ecological interactions, including the unique ‘Steely’ enzymes, consisting of a fusion between a fatty acid synthase and a chalcone synthase. We report here that D. discoideum further increases its polyketide potential by using the StlB Steely enzyme, and a downstream chlorinating enzyme, to make both a chlorinated signal molecule, DIF-1, during its multi-cellular development, and a set of abundant polyketides in terminally differentiated stalk cells. We identify one of these as a chlorinated dibenzofuran with potent anti-bacterial activity. To do this, StlB switches expression from prespore to stalk cells in late development and is cleaved to release the chalcone synthase domain. Expression of this domain alone in StlB null cells allows synthesis of the stalk-associated, chlorinated polyketides. Thus, by altered expression and processing of StlB, cells make first a signal molecule, and then abundant secondary metabolites, which we speculate help to protect the mature spores from bacterial infection.

Structure, development, and the salt response of salt bladders in Chenopodium album L.

Salt bladders are specialized epidermal structures that halophytes use to store and excrete excess salt. However, the cell wall composition during salt bladder development is unclear, and the functions of salt bladders in a few wild plants remain unexplored. Therefore, the present study examined salt bladder development, cell wall composition, and their roles under salt stress by employing bladder-brushed and unbrushed Chenopodium album plants. We found that the bladder cell of C. album was connected to the epidermal cells through a rectangular stalk cell and developed from the shoot tip and the young leaves. The polysaccharides of salt bladder cell wall showed dynamic distribution at different stages of development. Moreover, salt bladders affected Na+ and K+ accumulation, increased reactive oxygen species scavenging, and improved the osmoregulation and photosynthetic efficiency in leaves, subsequently enhancing the salt tolerance of plants. The findings strengthen our knowledge of the physiological mechanisms of the accessory structures in desert plants, which can be used as a reference for further research at the molecular level.

Autophagy of the somatic stalk cells nurses the propagating spores of Dictyostelid social amoebas

Background: Autophagy (self-feeding) assists survival of starving cells by partial self-digestion, while dormancy as cysts, spores or seeds enables long-term survival. Starving Dictyostelium amoebas construct multicellular fruiting bodies with spores and stalk cells, with many Dictyostelia still able to encyst individually like their single-celled ancestors. While autophagy mostly occurs in the somatic stalk cells, autophagy gene knock-outs in Dictyostelium discoideum (D. discoideum) formed no spores and lacked cAMP induction of prespore gene expression. Methods: To investigate whether autophagy also prevents encystation, we knocked-out autophagy genes atg5 and atg7 in the dictyostelid Polysphondylium pallidum, which forms both spores and cysts. We measured spore and cyst differentiation and viability in the knock-out as well as stalk and spore gene expression and its regulation by cAMP. We tested a hypothesis that spores require materials derived from autophagy in stalk cells. Sporulation requires secreted cAMP acting on receptors and intracellular cAMP acting on PKA. We compared the morphology and viability of spores developed in fruiting bodies with spores induced from single cells by stimulation with cAMP and 8Br-cAMP, a membrane-permeant PKA agonist. Results: Loss of autophagy in P. pallidum reduced but did not prevent encystation. However, spore, but not stalk differentiation, and cAMP-induced prespore gene expression were lost. Spores induced in vitro by cAMP and 8Br-cAMP were smaller and rounder than spores formed multicellularly and while they were not lysed by detergent they did not germinate, unlike multicellular spores. Conclusions: The stringent requirement of sporulation on both multicellularity and autophagy, which occurs mostly in stalk cells, suggests that stalk cells nurse the spores through autophagy. This highlights autophagy as a major cause for somatic cell evolution in early multicellularity.

Immunocytochemical Analysis of the Wall Ingrowths in the Digestive Gland Transfer Cells in Aldrovanda vesiculosa L. (Droseraceae).

Carnivorous plants are unique due to their ability to attract small animals or protozoa, retain them in specialized traps, digest them, and absorb nutrients from the dissolved prey material; however, to this end, these plants need a special secretion-digestive system (glands). A common trait of the digestive glands of carnivorous plants is the presence of transfer cells. Using the aquatic carnivorous species Aldrovanda vesiculosa, we showed carnivorous plants as a model for studies of wall ingrowths/transfer cells. We addressed the following questions: Is the cell wall ingrowth composition the same between carnivorous plant glands and other plant system models? Is there a difference in the cell wall ingrowth composition between various types of gland cells (glandular versus endodermoid cells)? Fluorescence microscopy and immunogold electron microscopy were employed to localize carbohydrate epitopes associated with major cell wall polysaccharides and glycoproteins. The cell wall ingrowths were enriched with arabinogalactan proteins (AGPs) localized with the JIM8, JIM13, and JIM14 epitopes. Both methylesterified and de-esterified homogalacturonans (HGs) were absent or weakly present in the wall ingrowths in transfer cells (stalk cells and head cells of the gland). Both the cell walls and the cell wall ingrowths in the transfer cells were rich in hemicelluloses: xyloglucan (LM15) and galactoxyloglucan (LM25). There were differences in the composition between the cell wall ingrowths and the primary cell walls in A. vesiculosa secretory gland cells in the case of the absence or inaccessibility of pectins (JIM5, LM19, JIM7, LM5, LM6 epitopes); thus, the wall ingrowths are specific cell wall microdomains. Even in the same organ (gland), transfer cells may differ in the composition of the cell wall ingrowths (glandular versus endodermoid cells). We found both similarities and differences in the composition of the cell wall ingrowths between the A. vesiculosa transfer cells and transfer cells of other plant species.