Dagmar Zeuschner

Immuno-electron tomography of ER exit sites reveals the existence of free COPII-coated transport carriers

Transport from the endoplasmic reticulum (ER) to the Golgi complex requires assembly of the COPII coat complex at ER exit sites. Recent studies have raised the question as to whether in mammalian cells COPII coats give rise to COPII-coated transport vesicles or instead form ER sub-domains that collect proteins for transport via non-coated carriers. To establish whether COPII-coated vesicles do exist in vivo, we developed approaches to combine quantitative immunogold labelling (to identify COPII) and three-dimensional electron tomography (to reconstruct entire membrane structures). In tomograms of both chemically fixed and high-pressure-frozen HepG2 cells, immuno-labelled COPII was found on ER-associated buds as well as on free ∼50-nm diameter vesicles. In addition, we identified a novel type of COPII-coated structure that consists of partially COPII-coated, 150–200-nm long, dumb-bell-shaped tubules. Both COPII-coated carriers also contain the SNARE protein Sec22b, which is necessary for downstream fusion events. Our studies unambiguously establish the existence of free, bona fide COPII-coated transport carriers at the ER–Golgi interface, suggesting that assembly of COPII coats in vivo can result in vesicle formation.

SNX1 defines an early endosomal recycling exit for sortilin and mannose 6-phosphate receptors

Mannose‐6‐phosphate receptors (MPRs) transport lysosomal hydrolases from the trans Golgi network (TGN) to endosomes. Recently, the multi‐ligand receptor sortilin has also been implicated in this transport, but the transport carriers involved herein have not been identified. By quantitative immuno‐electron microscopy, we localized endogenous sortilin of HepG2 cells predominantly to the TGN and endosomes. In the TGN, sortilin colocalized with MPRs in the same clathrin‐coated vesicles. In endosomes, sortilin and MPRs concentrated in sorting nexin 1 (SNX1)‐positive buds and vesicles. SNX1 depletion by small interfering RNA resulted in decreased pools of sortilin in the TGN and an increase in lysosomal degradation. These data indicate that sortilin and MPRs recycle to the TGN in SNX1‐dependent carriers, which we named endosome‐to‐TGN transport carriers (ETCs). Notably, ETCs emerge from early endosomes (EE), lack recycling plasma membrane proteins and by three‐dimensional electron tomography exhibit unique structural features. Hence, ETCs are distinct from hitherto described EE‐derived membranes involved in recycling. Our data emphasize an important role of EEs in recycling to the TGN and indicate that different, specialized exit events occur on the same EE vacuole.

Immunogold Labeling of Cryosections from High-Pressure Frozen Cells

Immunogold labeling of cryosections according to Tokuyasu (Tokuyasu KT. A technique for ultracyotomy of cell suspensions and tissues. J Cell Biol 1973;57:551–565), is an important and widely used method for immunoelectron microscopy. These sections are cut from material that is chemically fixed at room temperature (room temparature fixation, RTF). Lately in many morphological studies fast freezing followed by cryosubstitution fixation (CSF) is used instead of RTF. We have explored some new methods for applying immunogold labeling on cryosections from high‐pressure frozen cells (HepG2 cells, primary chondrocytes) and tissues (cartilage and exocrine pancreas). As immunolabeling has to be carried out on thawed and stable sections, we explored two ways to achieve this: (1) The section fixation method, as briefly reported before (Liou W et al. Histochem Cell Biol 1996;106:41–58 and Möbius W et al. J Histochem Cytochem 2002;50:43–55.) in which cryosections from freshly frozen cells were stabilized in mixtures of sucrose and methyl cellulose and varying concentrations of glutaraldehyde, formaldehyde and uranyl acetate (UA). Only occasionally does this method reveal section areas with excellent cell preservation and negatively stained membranes like Tokuyasu sections of RTF material. (Liou et al.) (2) The rehydration method, a novel approach, in which CSF with glutaraldehyde and/or osmium tetroxide (OsO4) was followed by rehydration and cryosectioning as in the Tokuyasu method. Especially, the addition of UA and low concentrations of water to the CSF medium favored superb membrane contrast. Immunogold labeling was as efficient as with the Tokuyasu method.

Identification of a clonally expanding haematopoietic compartment in bone marrow.

In mammals, postnatal haematopoiesis occurs in the bone marrow (BM) and involves specialized microenvironments controlling haematopoietic stem cell (HSC) behaviour and, in particular, stem cell dormancy and self‐renewal. While these processes have been linked to a number of different stromal cell types and signalling pathways, it is currently unclear whether BM has a homogenous architecture devoid of structural and functional partitions. Here, we show with genetic labelling techniques, high‐resolution imaging and functional experiments in mice that the periphery of the adult BM cavity harbours previously unrecognized compartments with distinct properties. These units, which we have termed hemospheres, were composed of endothelial, haematopoietic and mesenchymal cells, were enriched in CD150+ CD48− putative HSCs, and enabled rapid haematopoietic cell proliferation and clonal expansion. Inducible gene targeting of the receptor tyrosine kinase VEGFR2 in endothelial cells disrupted hemospheres and, concomitantly, reduced the number of CD150+ CD48− cells. Our results identify a previously unrecognized, vessel‐associated BM compartment with a specific localization and properties distinct from the marrow cavity.

Smed-SmB, a member of the LSm protein superfamily, is essential for chromatoid body organization and planarian stem cell proliferation

Planarians are an ideal model system to study in vivo the dynamics of adult pluripotent stem cells. However, our knowledge of the factors necessary for regulating the ‘stemness’ of the neoblasts, the adult stem cells of planarians, is sparse. Here, we report on the characterization of the first planarian member of the LSm protein superfamily, Smed-SmB, which is expressed in stem cells and neurons in Schmidtea mediterranea. LSm proteins are highly conserved key players of the splicing machinery. Our study shows that Smed-SmB protein, which is localized in the nucleus and the chromatoid body of stem cells, is required to safeguard the proliferative ability of the neoblasts. The chromatoid body, a cytoplasmatic ribonucleoprotein complex, is an essential regulator of the RNA metabolism required for the maintenance of metazoan germ cells. However, planarian neoblasts and neurons also rely on its functions. Remarkably, Smed-SmB dsRNA-mediated knockdown results in a rapid loss of organization of the chromatoid body, an impairment of the ability to post-transcriptionally process the transcripts of Smed-CycB, and a severe proliferative failure of the neoblasts. This chain of events leads to a quick depletion of the neoblast pool, resulting in a lethal phenotype for both regenerating and intact animals. In summary, our results suggest that Smed-SmB is an essential component of the chromatoid body, crucial to ensure a proper RNA metabolism and essential for stem cell proliferation.

CD99 and CD99L2 act at the same site as, but independently of, PECAM-1 during leukocyte diapedesis

Leukocyte extravasation depends on various adhesion receptors at endothelial cell contacts. Here we have analyzed how mouse CD99 and CD99L2 cooperate with PECAM-1. We found that antibodies against mouse CD99 and PECAM-1 trap neutrophils between endothelial cells in in vitro transmigration assays. A sequential function, as has been suggested for human PECAM-1 and CD99, could not be demonstrated. In contrast to these in vitro results, blocking CD99 or CD99L2 or gene disruption of PECAM-1 trapped neutrophils in vivo between endothelial cells and the underlying basement membrane as revealed by electron microscopy and by 3-dimensional confocal fluorescence microscopy in the inflamed cremaster tissue. Leukocyte extravasation was inhibited in interleukin-1beta-inflamed peritoneum and in the cremaster by PECAM-1 gene disruption and was further attenuated by blocking antibodies against CD99 and CD99L2. In addition, CD99 and CD99L2 were required for leukocyte extravasation in the cremaster after stimulation with tumor necrosis factor-alpha, where the need for PECAM-1 is known to be bypassed. We conclude that CD99 and CD99L2 act independently of PECAM-1 in leukocyte extravasation and cooperate in an independent way to help neutrophils overcome the endothelial basement membrane.

Integrin β1 controls VE-cadherin localization and blood vessel stability.

Angiogenic blood vessel growth requires several distinct but integrated cellular activities. Endothelial cell sprouting and proliferation lead to the expansion of the vasculature and give rise to a highly branched, immature plexus, which is subsequently reorganized into a mature and stable network. Although it is known that integrin-mediated cell-matrix interactions are indispensable for embryonic angiogenesis, little is known about the function of integrins in different steps of vascular morphogenesis. Here, by investigating the integrin β1-subunit with inducible and endothelial-specific gene targeting in the postnatal mouse retina, we show that β1 integrin promotes endothelial sprouting but is a negative regulator of proliferation. In maturing vessels, integrin β1 is indispensable for proper localization of VE-cadherin and thereby cell-cell junction integrity. The sum of our findings establishes that integrin β1 has critical functions in the growing and maturing vasculature, and is required for the formation of stable, non-leaky blood vessels.

The zebrafish common cardinal veins develop by a novel mechanism: lumen ensheathment

The formation and lumenization of blood vessels has been studied in some detail, but there is little understanding of the morphogenetic mechanisms by which endothelial cells (ECs) forming large caliber vessels aggregate, align themselves and finally form a lumen that can support blood flow. Here, we focus on the development of the zebrafish common cardinal veins (CCVs), which collect all the blood from the embryo and transport it back to the heart. We show that the angioblasts that eventually form the definitive CCVs become specified as a separate population distinct from the angioblasts that form the lateral dorsal aortae. The subsequent development of the CCVs represents a novel mechanism of vessel formation, during which the ECs delaminate and align along the inner surface of an existing luminal space. Thereby, the CCVs are initially established as open-ended endothelial tubes, which extend as single EC sheets along the flow routes of the circulating blood and eventually enclose the entire lumen in a process that we term ‘lumen ensheathment’. Furthermore, we found that the initial delamination of the ECs as well as the directional migration within the EC sheet depend on Cadherin 5 function. By contrast, EC proliferation within the growing CCV is controlled by Vascular endothelial growth factor C, which is provided by circulating erythrocytes. Our findings not only identify a novel mechanism of vascular lumen formation, but also suggest a new form of developmental crosstalk between hematopoietic and endothelial cell lineages.

ACBD5 and VAPB mediate membrane associations between peroxisomes and the ER

Peroxisomes (POs) and the endoplasmic reticulum (ER) cooperate in cellular lipid metabolism and form tight structural associations, which were first observed in ultrastructural studies decades ago. PO–ER associations have been suggested to impact on a diverse number of physiological processes, including lipid metabolism, phospholipid exchange, metabolite transport, signaling, and PO biogenesis. Despite their fundamental importance to cell metabolism, the mechanisms by which regions of the ER become tethered to POs are unknown, in particular in mammalian cells. Here, we identify the PO membrane protein acyl-coenzyme A–binding domain protein 5 (ACBD5) as a binding partner for the resident ER protein vesicle-associated membrane protein-associated protein B (VAPB). We show that ACBD5–VAPB interaction regulates PO–ER associations. Moreover, we demonstrate that loss of PO–ER association perturbs PO membrane expansion and increases PO movement. Our findings reveal the first molecular mechanism for establishing PO–ER associations in mammalian cells and report a new function for ACBD5 in PO–ER tethering.

Annexin A8 Regulates Late Endosome Organization and Function

Different classes of endosomes exhibit a characteristic intracellular steady-state distribution governed by interactions with the cytoskeleton. Late endosomes, organelles of the degradative lysosomal route, seem to require associated actin filaments for proper localization and function. We show here that the F-actin and phospholipid binding protein annexin A8 is associated specifically with late endosomes. Altering intracellular annexin A8 levels drastically affected the morphology and intracellular distribution of late endosomes. Trafficking through the degradative pathway was delayed in the absence of annexin A8, resulting in attenuated ligand-induced degradation of the epidermal growth factor receptor and prolonged epidermal growth factor-induced activation of mitogen-activated protein kinase. Depletion of annexin A8 reduced the association of late endosomal membranes with actin filaments. These results indicate that the defective cargo transport through the late endocytic pathway and the imbalanced signaling of activated receptors observed in the absence of annexin A8 results from the disturbed association of late endosomal membranes with the actin network, resulting in impaired actin-based late endosome motility.