Fat causes necrosis and inflammation in parenchymal cells in human steatotic liver

Adapted fixation methods for electron microscopy allowed us to study liver cell fine structure in 217 biopsies of intact human livers over the course of 10 years. The following novel observations and concepts arose: single fat droplets in parenchymal cells can grow to a volume four times larger than the original cell, thereby extremely marginalizing the cytoplasm with all organelles. Necrosis of single parenchymal cells, still containing one huge fat droplet, suggests death by fat in a process of single-cell steatonecrosis. In a later stage of single-cell steatonecrosis, neutrophils and erythrocytes surround the single fat droplet, forming an inflammatory fat follicle indicating the apparent onset of inflammation. Also, fat droplets frequently incorporate masses of filamentous fragments and other material, most probably representing Mallory substance. No other structure or material was found that could possibly represent Mallory bodies. We regularly observe the extrusion of huge fat droplets, traversing the peripheral cytoplasm of parenchymal cells, the Disse space and the endothelium. These fat droplets fill the sinusoid as a sinusoidal lipid embolus. In conclusion, adapted methods of fixation applied to human liver tissue revealed that single, huge fat droplets cause necrosis and inflammation in single parenchymal cells. Fat droplets also collect Mallory substance and give rise to sinusoidal fat emboli. Therefore, degreasing of the liver seems to be an essential therapeutic first step in the self-repairing of non-alcoholic fatty liver disease. This might directly reduce single-cell steatotic necrosis and inflammation as elements in non-alcoholic steatohepatitis progression.

Persistent growth of microtubules at low density

Microtubules (MTs) often form a polarized array with minus-ends anchored at the centrosome and plus-ends extended towards the cell margins. Plus-ends display behavior known as dynamic instability, – transitions between rapid shortening and slow growth. It is known that dynamic instability is regulated locally to ensure entry of MTs into nascent areas of cytoplasm, but details of this regulation remain largely unknown. Here, we test alternative hypothesis for the local regulation of MT behavior. We used microsurgery to isolate a portion of peripheral cytoplasm from MTs growing from the centrosome, crating cytoplasmic areas locally depleted of MTs. We found that in sparsely populated areas MT plus-ends persistently grew or paused but never shortened. In contrast, plus-ends that entered regions of cytoplasm densely populated with MTs frequently transitioned to shortening. Persistent growth of MTs in sparsely populated areas could not be explained by a local increase in concentration of free tubulin subunits or elevation of Rac1 activity proposed to enhance MT growth at the cell leading edge during locomotion. These observations suggest the existence of a MT-density dependent mechanism regulating MT dynamics that determines dynamic instability of MTs in densely populated areas of the cytoplasm and persistent growth in sparsely populated areas. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

AP-4 mediates export of ATG9A from the trans-Golgi network to promote autophagosome formation

AP-4 is a member of the heterotetrameric adaptor protein (AP) complex family involved in protein sorting in the endomembrane system of eukaryotic cells. Interest in AP-4 has recently risen with the discovery that mutations in any of its four subunits cause a form of hereditary spastic paraplegia (HSP) with intellectual disability. The critical sorting events mediated by AP-4 and the pathogenesis of AP-4 deficiency, however, remain poorly understood. Here we report the identification of ATG9A, the only multispanning membrane component of the core autophagy machinery, as a specific AP-4 cargo. AP-4 promotes signal-mediated export of ATG9A from the trans-Golgi network to the peripheral cytoplasm, contributing to lipidation of the autophagy protein LC3B and maturation of preautophagosomal structures. These findings implicate AP-4 as a regulator of autophagy and altered autophagy as a possible defect in AP-4–deficient HSP.

Biological Description of 109 Cases of Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN) from the French Network of BPDCN

Blastic plasmacytoid dendritic cell neoplasm is a clonal disease derived from precursors of plasmacytoid dendritic cells (pDC). It is a rare neoplasm involving the skin which may or may not be associated from the outset with a leukemic component. The disease invariably progresses to aggressive leukemic dissemination, leading to a differential diagnosis with acute leukemia. In 2004, we set up a French network to recruit biological data at diagnosis. Diagnosis was according to recommendations (Swerdlow et al, 2008), with, in addition, a mandatory panel of pDC markers (Garnache-Ottou et al, 2009) detected by flow cytometry or by immunohistochemistry on infiltrated blood, bone marrow or cutaneous lesions. In total, 109 cases of BPDCN were included in 35 hospitals (2000-2013). BPDCN is more prevalent in men (sex ratio 4.4/1) and in elderly subjects (median age: 63 years; 7 patients were <20 yo).S kin lesions are very prevalent (85%) with variable lesion types. Blood cell counts show variable leukocytosis (figure 1A) with presence of blasts in 65% of cases. Anemia and thrombopenia were present in 59% and 76% of cases respectively. Bone marrow aspiration showed blastic infiltration in 94% of cases. Indeed, in 7 cases, there was isolated cutaneous involvement at diagnosis, with neither blood nor bone marrow infiltration. Morphologies of blast cells were heterogeneous. Typical morphologies were the most frequent, including medium-sized cells with a blastic round or irregular nucleus, cytoplasm displayed faint and irregular basophilia and no granulation. In a contingent of the blastic populations, we observed small vacuoles in a peculiar arrangement under the cytoplasmic membrane (42%) or the presence of large pseudopodia (28%) or both (17%) (Figure 1B, C, D). Some cases showed a more immature morphology with larger cells, higher nucleo-cytoplasmic ratio, very visible nucleoli and reinforced basophilia (28%) or a pseudomonoblastic morphology (5%) (Figure 1E). Rare cases presented a pseudolymphocytic form (7%, figure 1F) or large granulations in the cytoplasm (2%). Peroxidase and esterase were negative in all cases. Dysplasia of hematopoietic lineages was observed in 29% (figure 1G). For 8% of patients, myelodysplastic syndrome was diagnosed before the diagnosis of BPDCN. Immunophenotype showed that HLA-DR and CD4 were expressed in all cases, but 4 cases did not express CD56 (confirmed using 3 different antibodies). Expression of markers of others hematopoietic lineages was frequent. Among myeloid markers, the most frequent was CD33 (46%), followed by CD117 (23%), whereas CD13, CD11c, CD15 and CD65 were rarely expressed. Monocyte markers (CD14, CD64, CD11b) and myeloperoxidase were never expressed. For the T lineage, CD2 and CD7 were the most frequent (62% and 58% respectively) whereas CD5 was rare (7%). No cytoplasmic or surface CD3 were detected. For the B lineage, CD22 was expressed in 16%, and low levels of cCD79a in 5%. Both were never expressed together, and no CD19, CD20 and immunoglobulins were found. Generally, we observed one of these antigens (Ags) per case, but in 44% of cases, there was a combination of 2 or 3 Ags from 2 or 3 different lineages. Immature Ags such as CD34 and CD133 were never found, and Tdt was found in 14% of cases. Cytogenetic analysis revealed abnormal caryotype in 65% of the 78 caryotypes evaluated, with 20 cases having a complex caryotype. The frequency of the chromosomal abnormalities involved are shown in Figure 1H. In conclusion, we describe the largest series of BPDCN to date in the literature. Detailed clinical and biological data at presentation allow improved recognition of this rare form of acute and aggressive leukemia, enabling early initiation of appropriate management. Figure 1. A: Blood cell count in 109 BPDCN patients at diagnosis. Bars represent the median. B: Typical BPDCN morphology. C: In this case, the nuclei were peripheral, cytoplasm presented heterogenous basophilia, vacuoles were rare but large pseudopodia are frequent. D: Typical morphology with frequent microvacuoles under the cytoplasmic membrane. E: Immature morphology. F: Pseudolymphocytic morphology. G: Presence of dysplasia in myeloid cells with Auer Rods in the granulocytes. The morphology of the Blastic cells is typical. H. Chromosomal abnormalities in 78 caryotypes evaluated: The histogram represents the number of cases in which each chromosome was involved (deletion, gain, translocations). Figure 1. A: Blood cell count in 109 BPDCN patients at diagnosis. Bars represent the median. B: Typical BPDCN morphology. C: In this case, the nuclei were peripheral, cytoplasm presented heterogenous basophilia, vacuoles were rare but large pseudopodia are frequent. D: Typical morphology with frequent microvacuoles under the cytoplasmic membrane. E: Immature morphology. F: Pseudolymphocytic morphology. G: Presence of dysplasia in myeloid cells with Auer Rods in the granulocytes. The morphology of the Blastic cells is typical. H. Chromosomal abnormalities in 78 caryotypes evaluated: The histogram represents the number of cases in which each chromosome was involved (deletion, gain, translocations). Disclosures Bardet: Celgene: Research Funding. Deconinck:CHUGAI: Other: Travel for international congress; PFIZER: Research Funding; ROCHE: Research Funding; NOVARTIS: Other: Travel for international congress; ALEXION: Other: Travel for international congress; JANSSEN: Other: Travel for international congress; LFB loboratory: Consultancy.

Compartmentalization in Penicillin G Biosynthesis by Penicillium chrysogenum PQ-96

The arrangement of organelles in the sub-apical productive non-growing vacuolated hyphal cells of the high- and the low-penicillin-pro- ducing strains Penicillium chrysogenum was compared using transmission electron microscopy. In the productive cells of the high-yielding strain the endoplasmic reticulum and the polyribosomes with associated peroxisomes are frequently arranged at the periphery of the cytoplasm and around the vacuoles. At the high activity of penicillin G biosynthesis the immuno-label of the cytosolic isopenicillin N synthase is concentrated at the polyribosomes arranged in the peripheral cytoplasm and along the tonoplast as well as around the peroxisomes. On the basis of the obtained results the compartmentalization of the pathway of penicillin G biosymthesis is discussed. The obtained results support the phenylacetic acid detoxification hypothesis of penicillin G biosynthesis.

Ultrastructural study of ectomycorrhizas on Pinus caribaea Morelet. var. hondurensis Barr. & Golf. seedlings

The ultrastructure of ectomycorrhizas formed between Pinus caribaea var. hondurensis inoculated with Pisolithus tinctorius (Pers.) Coker & Couch and Telephora terrestris (Ehrenb.) Fr. was analyzed just before the transplant of these seedlings to the field to ascertain if fungi are established in the roots. Ectomycorrhizal fungi formed a well-developed compact mantle in lateral roots. Vacuoles, nuclei and dolipore septa were observed in mantle hyphae and numerous nuclei, endoplasmatic reticulum and polymorphic mitochondria were frequently located in the cytoplasm of Hartig net hyphae adjacent to plant cortical cells. Highly vacuolated cortical cells contained droplets of electron-dense material, nucleus and some organelles were observed in a narrow region of peripheral cytoplasm. The ectomycorrhizas of P. caribaea var. hondurensis exhibited typical ultrastructural characteristics of a compatible and physiological active association.

Reorganization of the microtubule array in prophase/prometaphase requires cytoplasmic dynein-dependent microtubule transport

When mammalian somatic cells enter mitosis, a fundamental reorganization of the Mt cytoskeleton occurs that is characterized by the loss of the extensive interphase Mt array and the formation of a bipolar mitotic spindle. Microtubules in cells stably expressing GFP–α-tubulin were directly observed from prophase to just after nuclear envelope breakdown (NEBD) in early prometaphase. Our results demonstrate a transient stimulation of individual Mt dynamic turnover and the formation and inward motion of microtubule bundles in these cells. Motion of microtubule bundles was inhibited after antibody-mediated inhibition of cytoplasmic dynein/dynactin, but was not inhibited after inhibition of the kinesin-related motor Eg5 or myosin II. In metaphase cells, assembly of small foci of Mts was detected at sites distant from the spindle; these Mts were also moved inward. We propose that cytoplasmic dynein-dependent inward motion of Mts functions to remove Mts from the cytoplasm at prophase and from the peripheral cytoplasm through metaphase. The data demonstrate that dynamic astral Mts search the cytoplasm for other Mts, as well as chromosomes, in mitotic cells.

Ultrastructure of spermatogenesis in the free-living marine nematode Pontonema vulgare (Enoplida, Oncholaimidae)

Spermatogenesis in testes of the free-living marine nematode Pontonema vulgare was studied with electron microscopy. The nucleus of spermatocytes has a large nucleolus; the cytoplasm is filled with numerous ribosomes, mitochondria, cisternae of the rough endoplasmic reticulum (RER), Golgi bodies, and flattened osmiophilic cisternae, which are interpreted as the modified membranous organelles (MO) of the spermatozoa of other nematodes studied. After completion of the second meiotic telophase, the nucleus is surrounded by a newly formed nuclear envelope. Further nucleus transformation includes condensation of the chromatin and shrinkage of the nuclear envelope. The deep infoldings of the nuclear envelope give a starlike shape to the nuclei. The cytoplasm of the early spermatids contains the same organelles as in the late spermatocytes, including MO. Many of the latter assume a cuplike or pocketlike shape. During spermatogenesis the peripheral cytoplasm containing the ribosomes, RER, Golgi bodies, and transparent vesicles moves to one pole of the cell forming the residual body. The main cell body of the late spermatid includes the nucleus, mitochondria, and MO embedded in a dense filamentous matrix. The fibrous bodies (FB) of a paracrystalline structure occur in spermatids throughout their developmental transformations. The central part of the spermatozoa contains a starlike nucleus with a nuclear envelope. The filamentous cytoplasm of the spermatozoa includes mitochondria and MO. The spermatozoa extracted from the testes form numerous long filopodia. The dense filamentous cytoplasm of the spermatozoa is continuous with the content of the filipodia. The reconstitution of the nuclear envelope and separate development of MO and FB described in P. vulgare spermatogenesis are the special characters of enoplid nematodes. The reduced character of FB development and simplified structure of MO differentiate P. vulgare from other nematodes studied.

Transferrin receptor recycling in the absence of perinuclear recycling endosomes

In mammalian cells, internalized receptors such as transferrin (Tfn) receptor are presumed to pass sequentially through early endosomes (EEs) and perinuclear recycling endosomes (REs) before returning to the plasma membrane. Whether passage through RE is obligatory, however, remains unclear. Kinetic analysis of endocytosis in CHO cells suggested that the majority of internalized Tfn bypassed REs returning to the surface from EEs. To determine directly if REs are dispensable for recycling, we studied Tfn recycling in cytoplasts microsurgically created to contain peripheral EEs but to exclude perinuclear REs. The cytoplasts actively internalized and recycled Tfn. Surprisingly, they also exhibited spatially and temporally distinct endosome populations. The first appeared to correspond to EEs, labeling initially with Tfn, being positive for early endosomal antigen 1 (EEA-1) and containing only small amounts of Rab11, an RE marker. The second was EEA-1 negative and with time recruited Rab11, suggesting that cytoplasts assembled functional REs. These results suggest that although perinuclear REs are not essential components of the Tfn recycling pathway, they are dynamic structures which preexist in the peripheral cytoplasm or can be regenerated from EE- and cytosol-derived components such as Rab11.