Tubular Organelles Research Articles

Branching Off: New Insight Into Lysosomes as Tubular Organelles.

Lysosomes are acidic, membrane-bound organelles that play essential roles in cellular quality control, metabolism, and signaling. The lysosomes of a cell are commonly depicted as vesicular organelles. Yet, lysosomes in fact show a high degree of ultrastructural heterogeneity. In some biological contexts, lysosome membranes naturally transform into tubular, non-vesicular morphologies. Though the purpose and regulation of tubular lysosomes has been historically understudied, emerging evidence suggests that tubular lysosomes may carry out unique activities, both degradative and non-degradative, that are critical to cell behavior, function, and viability. Here, we discuss recent advances in understanding the biological significance of tubular lysosomes in cellular physiology, and we highlight a growing number of examples that indicate the centrality of this special class of lysosomes to health and disease.

Mitochondrial Quality Control Governed by Ubiquitin.

Mitochondria are essential organelles important for energy production, proliferation, and cell death. Biogenesis, homeostasis, and degradation of this organelle are tightly controlled to match cellular needs and counteract chronic stress conditions. Despite providing their own DNA, the vast majority of mitochondrial proteins are encoded in the nucleus, synthesized by cytosolic ribosomes, and subsequently imported into different mitochondrial compartments. The integrity of the mitochondrial proteome is permanently challenged by defects in folding, transport, and turnover of mitochondrial proteins. Therefore, damaged proteins are constantly sequestered from the outer mitochondrial membrane and targeted for proteasomal degradation in the cytosol via mitochondrial-associated degradation (MAD). Recent studies identified specialized quality control mechanisms important to decrease mislocalized proteins, which affect the mitochondrial import machinery. Interestingly, central factors of these ubiquitin-dependent pathways are shared with the ER-associated degradation (ERAD) machinery, indicating close collaboration between both tubular organelles. Here, we summarize recently described cellular stress response mechanisms, which are triggered by defects in mitochondrial protein import and quality control. Moreover, we discuss how ubiquitin-dependent degradation is integrated with cytosolic stress responses, particularly focused on the crosstalk between MAD and ERAD.

Impact of global structure on diffusive exploration of organelle networks

We investigate diffusive search on planar networks, motivated by tubular organelle networks in cell biology that contain molecules searching for reaction partners and binding sites. Exact calculation of the diffusive mean first-passage time on a spatial network is used to characterize the typical search time as a function of network connectivity. We find that global structural properties — the total edge length and number of loops — are sufficient to largely determine network exploration times for a variety of both synthetic planar networks and organelle morphologies extracted from living cells. For synthetic networks on a lattice, we predict the search time dependence on these global structural parameters by connecting with percolation theory, providing a bridge from irregular real-world networks to a simpler physical model. The dependence of search time on global network structural properties suggests that network architecture can be designed for efficient search without controlling the precise arrangement of connections. Specifically, increasing the number of loops substantially decreases search times, pointing to a potential physical mechanism for regulating reaction rates within organelle network structures.

Identification of a New Exo-Endocytic Mechanism Triggered by Corticotropin-Releasing Hormone in Mast Cells.

The key role of mast cells (MC), either in development of inflammatory pathologies or in response to environmental stress, has been widely reported in recent years. Previous studies have described the effects of corticotropin-releasing hormone (CRH), which is released from inflamed tissues by cellular stress signals, on MC degranulation, a process possibly driven by selective secretion of mediators (piecemeal degranulation). In this study, we introduce a novel granular exo-endocytic pathway induced by CRH on peritoneal MC. We found that CRH triggers substantial exocytosis, which is even stronger than that induced by Ag stimulation and is characterized by large quantal size release events. Membrane fluorescence increases during stimulation in the presence of FM1-43 dye, corroborating the strength of this exocytosis, given that discrete upward fluorescence steps are often observed and suggesting that secretory granules are preferentially released by compound exocytosis. Additionally, the presence of a depot of large tubular organelles in the cytoplasm suggests that the exocytotic process is tightly coupled to a fast compound endocytosis. This CRH-stimulated mechanism is mediated through activation of adenylate cyclase and an increase of cAMP and intracellular Ca(2+), as evidenced by the fact that the effect of CRH is mimicked by forskolin and 8-bromo-cAMP. Thus, these outcomes constitute new evidence for the critical role of MC in pathophysiological conditions within a cellular stress environment and an alternative membrane trafficking route mediated by CRH.

Tomographic reconstruction reveals the morphology of a unique cellular organelle, the aggregated macrotubules (Macrotubuli aggregati) of human retinal horizontal cells.

Horizontal cells of the human retina contain unique tubular organelles that have a diameter which is about 10 times larger than that of microtubules (~230 nm). These macrotubuli in most cases form regular aggregates. Therefore we propose to introduce them as Macrotubuli aggregati in the Terminologia histologica. Tomographic investigation of the structures revealed that the walls of the tubules most probably consist of intermediate filaments running nearly parallel to each other and show somewhat regularly attached ribosomes on their inner and also outer surface. About 2% of the organelles exhibit double- to multiple layered walls and less than 1% resemble large scrolls. The tubules may extend 10 to over 20 μm in the cytoplasm and are also encountered in soma-near processes extending into the outer plexiform layer. It remains unclear why these structures are only present in humans and few other species and why almost only in horizontal cells. Speculations on possible functions are discussed.

Scanning Fluorescence Correlation Spectroscopy in Mitochondria of Living Cells

New methods to quantify dynamics and interactions of intracellular species provide key insights in cell biology. Fluorescence Correlation Spectroscopy (FCS) utilizes temporal autocorrelation of fluorescence fluctuations to study the dynamic properties of labeled molecules. Previous studies characterize diffusion and interaction of proteins in the nucleus and cytosol using FCS. However, only few deal with tubular organelles like the endoplasmic reticulum and mitochondria. The ability to accurately place the confocal volume in these dynamic organelles limits point FCS in vivo. Originally applied in membranes, scanning FCS (SFCS) addresses these challenges.We applied SFCS to measure concentration and dynamic properties fluorescently-labeled molecules in mitochondria. SFCS accurately positions the confocal volume (in the x-y plane) by moving it along a linear scanning path. At a scanning orientation perpendicular to the mitochondria, we can also reduce photobleaching due to the brief residence times in the confocal volume.General scheme of SFCS. (a) shows a fluorescence image of a mitochondria. The scan direction is oriented perpendicular to the mitochondria. (b) shows the acquired data before selection of mitochondria(orange box) and alignment(c). (d) represents the fluctuation trace, which is correlated and fitted to diffusion models (e).View Large Image | View Hi-Res Image | Download PowerPoint Slide

Kupffer cell structure in the juvenile Nile crocodile,Crocodylus niloticus

The morphology of Kupffer cells was examined in the liver of the juvenile Nile crocodile using light microscopy and transmission electron microscopy. Pleomorphic Kupffer cells were located in the sinusoids, in the space of Disse, in the hepatic parenchyma and often connected adjacent sinusoids. The cell surfaces were irregular due to the presence of filopodia and lamelliapodia with phagocytosis of white blood cells, red blood cells and thrombocytes being evident. The cells were in close contact with endothelial cells and pit cells in the sinusoidal lumen and with stellate cells in the space of Disse. The cytoplasm contained large phagosomes comprising a combination of ceroid pigment, melanosomes and siderosomes. The nuclei were often indented and eccentrically placed due to the presence of the phagosomes. Conspicuous clusters of membrane-bound tubular organelles with a filamentous or crystalline interior were observed in the cytoplasm. The clusters were sometimes separated into smaller groups around phagosomes. A clear zone existed between the limiting membrane and the interior of these tubular organelles with the electron-dense interior profiles being, respectively, circular, angular or divided. The tubular organelles have not previously been described in Kupffer cells and possibly represent lysosomes with specialized functions. Mitochondria, microtubules, Golgi profiles, granular and smooth endoplasmic reticulum, and a few cytoplasmic lipid droplets were also present. The presence of the tubular organelles and the occurrence of the Kupffer cells in different locations in the liver of the juvenile Nile crocodile are indicative of particularly active and mobile cells.

The platelet interior revisited: electron tomography reveals tubular α-granule subtypes

AbstractWe have used (cryo) electron tomography to provide a 3-dimensional (3D) map of the intracellular membrane organization of human platelets at high spatial resolution. Our study shows that the open canalicular system and dense tubular system are highly intertwined and form close associations in specialized membrane regions. 3D reconstructions of individual α-granules revealed large heterogeneity in their membrane organization. On the basis of their divergent morphology, we categorized α-granules into the following subtypes: spherical granules with electron-dense and electron-lucent zone containing 12-nm von Willebrand factor tubules, subtypes containing a multitude of luminal vesicles, 50-nm-wide tubular organelles, and a population with 18.4-nm crystalline cross-striations. Low-dose (cryo) electron tomography and 3D reconstruction of whole vitrified platelets confirmed the existence of long tubular granules with a remarkably curved architecture. Immunoelectron microscopy confirmed that these extended structures represent α-granule subtypes. Tubular α-granules represent approximately 16% of the total α-granule population and are detected in approximately half of the platelet population. They express membrane-bound proteins GLUT3 and αIIb-β3 integrin and contain abundant fibrinogen and albumin but low levels of β-thromboglobulin and no von Willebrand factor. Our 3D study demonstrates that, besides the existence of morphologically different α-granule subtypes, high spatial segregation of cargo exists within individual α-granules.

Enigmatic, ultrasmall, uncultivated Archaea.

Metagenomics has provided access to genomes of as yet uncultivated microorganisms in natural environments, yet there are gaps in our knowledge-particularly for Archaea-that occur at relatively low abundance and in extreme environments. Ultrasmall cells (<500 nm in diameter) from lineages without cultivated representatives that branch near the crenarchaeal/euryarchaeal divide have been detected in a variety of acidic ecosystems. We reconstructed composite, near-complete approximately 1-Mb genomes for three lineages, referred to as ARMAN (archaeal Richmond Mine acidophilic nanoorganisms), from environmental samples and a biofilm filtrate. Genes of two lineages are among the smallest yet described, enabling a 10% higher coding density than found genomes of the same size, and there are noncontiguous genes. No biological function could be inferred for up to 45% of genes and no more than 63% of the predicted proteins could be assigned to a revised set of archaeal clusters of orthologous groups. Some core metabolic genes are more common in Crenarchaeota than Euryarchaeota, up to 21% of genes have the highest sequence identity to bacterial genes, and 12 belong to clusters of orthologous groups that were previously exclusive to bacteria. A small subset of 3D cryo-electron tomographic reconstructions clearly show penetration of the ARMAN cell wall and cytoplasmic membranes by protuberances extended from cells of the archaeal order Thermoplasmatales. Interspecies interactions, the presence of a unique internal tubular organelle [Comolli, et al. (2009) ISME J 3:159-167], and many genes previously only affiliated with Crenarchaea or Bacteria indicate extensive unique physiology in organisms that branched close to the time that Cren- and Euryarchaeotal lineages diverged.

Mitochondrial Cristae Revealed with Focused Light

Because of the diffraction resolution barrier, optical microscopes have so far failed in visualizing the mitochondrial cristae, that is, the folds of the inner membrane of this 200 to 400 nm diameter sized tubular organelle. Realizing a approximately 30 nm isotropic subdiffraction resolution in isoSTED fluorescence nanoscopy, we have visualized these essential structures in the mitochondria of intact cells. We find a pronounced heterogeneity in the cristae arrangements even within individual mitochondrial tubules.

Overexpression of a Mutant Form of EhRabA, a Unique Rab GTPase of Entamoeba histolytica , Alters Endoplasmic Reticulum Morphology and Localization of the Gal/GalNAc Adherence Lectin

Entamoeba histolytica is a protozoan parasite that causes amoebic dysentery and liver abscess. Vesicle trafficking events, such as phagocytosis and delivery of plasma membrane proteins, have been implicated in pathogenicity. Rab GTPases are proteins whose primary function is to regulate vesicle trafficking; therefore, understanding the function of Rabs in this organism may provide insight into virulence. E. histolytica possesses a number of unique Rabs that exhibit limited homology to host Rabs. In this study we examined the function of one such Rab, EhRabA, by characterizing a mutant overexpressing a constitutively GTP-bound version of the protein. Overexpression of mutant EhRabA resulted in decreased adhesion to and phagocytosis of human red blood cells and in the appearance of large tubular organelles that could be stained with endoplasmic reticulum (ER)-specific but not Golgi complex-specific antibodies. Consistent with the adhesion defect, two subunits of a cell surface adhesin, the galactose/N-acetylgalactosamine lectin, were mislocalized to the novel organelle. A cysteine protease, EhCP2, was also localized to the ER-like compartment in the mutant; however, the localization of two additional cell surface proteins, Igl and SREHP, remained unchanged in the mutant. The phenotype of the mutant could be recapitulated by treatment with brefeldin A, a cellular toxin that disrupts ER-to-Golgi apparatus vesicle traffic. This suggests that EhRabA influences vesicle trafficking pathways that are also sensitive to brefeldin A. Together, the data indicate that EhRabA directly or indirectly influences the morphology of secretory organelles and regulates trafficking of a subset of secretory proteins in E. histolytica.

Nitric oxide-induced mitochondrial fission is regulated by dynamin-related GTPases in neurons

Mitochondria are present as tubular organelles in neuronal projections. Here, we report that mitochondria undergo profound fission in response to nitric oxide (NO) in cortical neurons of primary cultures. Mitochondrial fission by NO occurs long before neurite injury and neuronal cell death. Furthermore, fission is accompanied by ultrastructural damage of mitochondria, autophagy, ATP decline and generation of free radicals. Fission is occasionally asymmetric and can be reversible. Strikingly, mitochondrial fission is also an early event in ischemic stroke in vivo. Mitofusin 1 (Mfn1) or dominant-negative Dynamin related protein 1 (Drp1(K38A)) inhibits mitochondrial fission induced by NO, rotenone and Amyloid-beta peptide. Conversely, overexpression of Drp1 or Fis1 elicits fission and increases neuronal loss. Importantly, NO-induced neuronal cell death was mitigated by Mfn1 and Drp1(K38A). Thus, persistent mitochondrial fission may play a causal role in NO-mediated neurotoxicity.

Ni2+ induces changes in the morphology of vacuoles, mitochondria and microtubules in Paxillus involutus cells

Organelles of ectomycorrhizal fungi are known to respond to changes in the extracellular environment. The response of vacuoles, mitochondria and microtubules to short-term nickel (Ni2+) exposure were investigated in hyphal tip cells of a Paxillus involutus from a heavy metal-rich soil. Vacuoles, mitochondria and microtubules were labelled with Oregon Green 488 carboxylic acid diacetate, 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)) and anti-alpha-tubulin antibodies, respectively; hyphae were treated with NiSO4 in the range of 0-1 mmol l(-1) and examined microscopically. Untreated hyphal tip cells contained tubular vacuole and mitochondrial networks. Ni2+ caused loss of organelle tubularity and severe microtubule disruption that were exposure-time and concentration dependent. Fine tubular vacuoles thickened and eventually became spherical in some hyphae, tubular mitochondria fragmented and microtubules shortened and aggregated into patches in most hyphae. Tubular vacuoles reformed on NiSO4 removal and tubular mitochondria in the presence of NiSO4 suggesting cellular detoxification. These results demonstrate that Ni2+ induces changes in organelle and microtubule morphology. Recovery of tubular organelles to pretreatment morphology after Ni2+ exposure suggests cellular detoxification of the metal ion.

Ni2+ induces changes in the morphology of vacuoles, mitochondria and microtubules in Paxillus involutus cells

Organelles of ectomycorrhizal fungi are known to respond to changes in the extracellular environment. The response of vacuoles, mitochondria and microtubules to short-term nickel (Ni2+) exposure were investigated in hyphal tip cells of a Paxillus involutus from a heavy metal-rich soil. Vacuoles, mitochondria and microtubules were labelled with Oregon Green 488 carboxylic acid diacetate, 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)) and anti-alpha-tubulin antibodies, respectively; hyphae were treated with NiSO4 in the range of 0-1 mmol l(-1) and examined microscopically. Untreated hyphal tip cells contained tubular vacuole and mitochondrial networks. Ni2+ caused loss of organelle tubularity and severe microtubule disruption that were exposure-time and concentration dependent. Fine tubular vacuoles thickened and eventually became spherical in some hyphae, tubular mitochondria fragmented and microtubules shortened and aggregated into patches in most hyphae. Tubular vacuoles reformed on NiSO4 removal and tubular mitochondria in the presence of NiSO4 suggesting cellular detoxification. These results demonstrate that Ni2+ induces changes in organelle and microtubule morphology. Recovery of tubular organelles to pretreatment morphology after Ni2+ exposure suggests cellular detoxification of the metal ion.

Dynamics of polymorphism of acidocalcisomes in Leishmania parasites.

Growth of Leishmania mexicana amazonensis promastigotes in different culture media resulted in structurally and chemically different acidocalcisomes. When grown in SDM-79 medium, the promastigotes showed large spherical acidocalcisomes of up to 1.2 microm diameter distributed throughout the cell. X-ray microanalysis and elemental mapping of the organelles showed large amounts of oxygen, phosphorus, sodium, potassium, magnesium, calcium, and zinc. Immunofluorescence microscopy using antisera raised against a peptide sequence of the vacuolar-type proton pyrophosphatase of Arabidopsis thaliana that is conserved in the Leishmania enzyme, indicated localization in acidocalcisomes. When cells were transferred to Warren's medium, the acidocalcisomes transformed from spherical into branched tubular organelles. The labeling pattern of the vacuolar proton-pyrophosphatase, considered as a marker for the organelle, changed accompanying the structural changes of the acidocalcisomes, and the enzyme showed an apparently lower proton-transporting activity when measured in digitonin-permeabilized promastigotes. X-ray microanalysis and elemental mapping of these structures revealed the additional presence of iron. Together, the results reveal that the morphology and composition of acidocalcisomes are greatly influenced by the culture conditions.

NANOJET as a chemical scalpel for accessing the internal 3D-structure of biological cells

The aim of this work is to obtain information about the internal structure of biological objects (BOs) using the Nanonozzle Plasma Jet Microfabrication Tool (NANOJET). As BOs we used Dictyostelium amoeba. Our results show that the NANOJET allows removal of the cell plasma membrane with negligible damaging its internal structures. Using scanning electron microscopy, we are able to see the spherical and tubular organelles within the cell.

Mgm1p, a dynamin-related GTPase, is essential for fusion of the mitochondrial outer membrane.

In Saccharomyces cerevisiae, mitochondrial fusion requires at least two outer membrane proteins, Fzo1p and Ugo1p. We provide direct evidence that the dynamin-related Mgm1 protein is also required for mitochondrial fusion. Like fzo1 and ugo1 mutants, cells disrupted for the MGM1 gene contain numerous mitochondrial fragments instead of the few long, tubular organelles seen in wild-type cells. Fragmentation of mitochondria in mgm1 mutants is rescued by disrupting DNM1, a gene required for mitochondrial division. In zygotes formed by mating mgm1 mutants, mitochondria do not fuse and mix their contents. Introducing mutations in the GTPase domain of Mgm1p completely block mitochondrial fusion. Furthermore, we show that mgm1 mutants fail to fuse both their mitochondrial outer and inner membranes. Electron microscopy demonstrates that although mgm1 mutants display aberrant mitochondrial inner membrane cristae, mgm1 dnm1 double mutants restore normal inner membrane structures. However, mgm1 dnm1 mutants remain defective in mitochondrial fusion, indicating that mitochondrial fusion requires Mgm1p regardless of the morphology of mitochondria. Finally, we find that Mgm1p, Fzo1p, and Ugo1p physically interact in the mitochondrial outer membrane. Our results raise the possibility that Mgm1p regulates fusion of the mitochondrial outer membrane through its interactions with Fzo1p and Ugo1p.

Formation and interaction of membrane tubes.

We show that the formation of membrane tubes (or membrane tethers), which is a crucial step in many biological processes, is highly nontrivial and involves first-order shape transitions. The force exerted by an emerging tube is a nonmonotonic function of its length. We point out that tubes attract each other, which eventually leads to their coalescence. We also show that detached tubes behave like semiflexible filaments with a rather short persistence length. We suggest that these properties play an important role in the formation and structure of tubular organelles.

Vacuoles and the fungal lifestyle

Summary The long tubular branching hypha is the most important fungal growth form, although some fungi grow as discrete spherical or ellipsoidal yeast cells. Vacuoles are ubiquitous in yeast cells as well as in hyphae of filamentous fungi and can be readily stained by simple methods of dye uptake. They appear as spherical or tubular organelles of extremely variable size. Vacuoles are known or suspected to play several crucial roles in the physiology of vegetative growth. Only some of these roles are related to the activity of lytic intravacuolar enzymes. Thus, vacuoles can degrade redundant organelles, presumably recycling macromolecules, especially under conditions of nutrient starvation. They are also the destination of endocytotic vesicles originating by inward budding of the plasma membrane, thereby providing a mechanism of ‘face-lifting’ of the plasma membrane and a means of communication with the extracellular environment. Further, vacuoles store organic and inorganic nutrients, and they may detoxify the cytoplasm by sequestration of toxic substances. Adjacent vacuoles are often linked to each other by fine channels through which stored nutrients can be transported over long distances by peristalsis. This may be of relevance especially in mycorrhizal fungi. In specialized fungal cells, vacuoles are probably involved in the synthesis of low-molecular weight compatible solutes. Their accumulation draws water into the cell and generates turgor pressure for infection by plant-pathogenic fungi or for active spore discharge mechanisms. In contrast, in wall-less developmental states such as zoospores, vacuoles serve to eliminate excess water by expulsion. It is likely that functionally different subpopulations of vacuoles exist in fungi to perform such a multitude of tasks, although experimental evidence for this is not yet available. The vacuolar system therefore presents a rewarding field for much-needed further study.

Reorganization of multivesicular bodies regulates MHC class II antigen presentation by dendritic cells.

Immature dendritic cells (DCs) sample their environment for antigens and after stimulation present peptide associated with major histocompatibility complex class II (MHC II) to naive T cells. We have studied the intracellular trafficking of MHC II in cultured DCs. In immature cells, the majority of MHC II was stored intracellularly at the internal vesicles of multivesicular bodies (MVBs). In contrast, DM, an accessory molecule required for peptide loading, was located predominantly at the limiting membrane of MVBs. After stimulation, the internal vesicles carrying MHC II were transferred to the limiting membrane of the MVB, bringing MHC II and DM to the same membrane domain. Concomitantly, the MVBs transformed into long tubular organelles that extended into the periphery of the cells. Vesicles that were formed at the tips of these tubules nonselectively incorporated MHC II and DM and presumably mediated transport to the plasma membrane. We propose that in maturing DCs, the reorganization of MVBs is fundamental for the timing of MHC II antigen loading and transport to the plasma membrane.