From fate to function: the Drosophila trachea and salivary gland as models for tubulogenesis

Tube formation is a ubiquitous process required to sustain life in multicellular organisms. The tubular organs of adult mammals include the lungs, vasculature, digestive and excretory systems, as well as secretory organs such as the pancreas, salivary, prostate, and mammary glands. Other tissues, including the embryonic heart and neural tube, have requisite stages of tubular organization early in development. To learn the molecular and cellular basis of how epithelial cells are organized into tubular organs of various shapes and sizes, investigators have focused on the Drosophila trachea and salivary gland as model genetic systems for branched and unbranched tubes, respectively. Both organs begin as polarized epithelial placodes, which through coordinated cell shape changes, cell rearrangement, and cell migration form elongated tubes. Here, we discuss what has been discovered regarding the details of cell fate specification and tube formation in the two organs; these discoveries reveal significant conservation in the cellular and molecular events of tubulogenesis.     

The luminal connection: From animal development to lumopathies

Interconnection of epithelial tubules is a crucial process during organogenesis. Organisms have evolved sets of molecular and cellular strategies to generate an interconnected tubular network during animal development. Spatiotemporal control of common cellular strategies includes dissolution of the basement membrane, apoptosis, rearrangements of cell adhesion junctions, and mesenchymal-like invasive cellular behaviors prior to tubular interconnection. Different model systems exhibit varying degrees of active invasive-like behaviors that precede tubular interconnection, which may reflect changes in cell polarity or differential adhesive cell states. Studies in this newly-emerging field of tubular interconnections will provide a greater understanding of pediatric diseases and cancer metastasis, as well as generate fundamentally new insights into lumen formation pathology, or lumopathies.     

Novel mechanisms of tube-size regulation revealed by the Drosophila trachea

The size of various tubes within tubular organs such as the lung, vascular system and kidney must be finely tuned for the optimal delivery of gases, nutrients, waste and cells within the entire organism. Aberrant tube sizes lead to devastating human illnesses, such as polycystic kidney disease, fibrocystic breast disease, pancreatic cystic neoplasm and thyroid nodules. However, the underlying mechanisms that are responsible for tube-size regulation have yet to be fully understood. Therefore, no effective treatments are available for disorders caused by tube-size defects. Recently, the Drosophila tracheal system has emerged as an excellent in vivo model to explore the fundamental mechanisms of tube-size regulation. Here, we discuss the role of the apical luminal matrix, cell polarity and signaling pathways in regulating tube size in Drosophila trachea. Previous studies of the Drosophila tracheal system have provided general insights into epithelial tube morphogenesis. Mechanisms that regulate tube size in Drosophila trachea could be well conserved in mammalian tubular organs. This knowledge should greatly aid our understanding of tubular organogenesis in vertebrates and potentially lead to new avenues for the treatment of human disease caused by tube-size defects.     

Drosophila tracheal morphogenesis: intricate cellular solutions to basic plumbing problems

The cellular architecture of tubular organs suggests striking similarities in the mechanisms of tubulogenesis between species. The formation of the Drosophila respiratory organ (trachea) highlights the basic principles of branch patterning and tube growth that generate a highly elaborate but stereotyped epithelial tubular network. Oriented cell migration, changes in cell shape, selective growth of the apical cell membrane and intracellular lumen formation are essential events in this process. These morphogenetic processes build four structurally distinct classes of tubes that facilitate optimal airflow and gas exchange with target tissues. The molecular players in these plots include attractant and repellent signals, differentiation factors that cause a high diversity of cell fates within the epithelium, and determinants of tube formation and dimensions.     

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific shapes. Drosophila tracheal terminal cells have become a powerful genetic model to identify and elucidate the roles of genes required for generating cellular morphologies. Terminal cells are a component of a branched tubular network, the tracheal system that functions to supply oxygen to internal tissues. Terminal cells are an excellent model for investigating questions of cell shape as they possess two distinct cellular architectures. First, terminal cells have an elaborate branched morphology, similar to complex neurons; second, terminal cell branches are formed as thin tubes and contain a membrane-bound intracellular lumen. Quantitative analysis of terminal cell branch number, branch organization and individual branch shape, can be used to provide information about the role of specific genetic mechanisms in the making of a branched cell. Analysis of tube formation in these cells can reveal conserved mechanisms of tubulogenesis common to other tubular networks, such as the vertebrate vasculature. Here we describe techniques that can be used to rapidly fix, image, and analyze both branching patterns and tube formation in terminal cells within Drosophila larvae. These techniques can be used to analyze terminal cells in wild-type and mutant animals, or genetic mosaics. Because of the high efficiency of this protocol, it is also well suited for genetic, RNAi-based, or drug screens in the Drosophila tracheal system.     

Tube morphogenesis: making and shaping biological tubes

Many organs are composed of epithelial tubes that transport vital fluids. Such tubular organs develop in many different ways and generate tubes of widely varying sizes and structures, but always with the apical epithelial surface lining the lumen. We describe recent progress in several diverse cell culture and genetic models of tube morphogenesis, which suggest apical membrane biogenesis, vesicle fusion, and secretion play central roles in tube formation and growth. We propose a unifying mechanism of tube morphogenesis that has been modified to create tube diversity and describe how defects in the tube size-sensing step can lead to polycystic kidney disease.     

Mechanism of Membranous Tunnelling Nanotube Formation in Viral Genome Delivery

In internal membrane-containing viruses, a lipid vesicle enclosed by the icosahedral capsid protects the genome. It has been postulated that this internal membrane is the genome delivery device of the virus. Viruses built with this architectural principle infect hosts in all three domains of cellular life. Here, using a combination of electron microscopy techniques, we investigate bacteriophage PRD1, the best understood model for such viruses, to unveil the mechanism behind the genome translocation across the cell envelope. To deliver its double-stranded DNA, the icosahedral protein-rich virus membrane transforms into a tubular structure protruding from one of the 12 vertices of the capsid. We suggest that this viral nanotube exits from the same vertex used for DNA packaging, which is biochemically distinct from the other 11. The tube crosses the capsid through an aperture corresponding to the loss of the peripentonal P3 major capsid protein trimers, penton protein P31 and membrane protein P16. The remodeling of the internal viral membrane is nucleated by changes in osmolarity and loss of capsid-membrane interactions as consequence of the de-capping of the vertices. This engages the polymerization of the tail tube, which is structured by membrane-associated proteins. We have observed that the proteo-lipidic tube in vivo can pierce the gram-negative bacterial cell envelope allowing the viral genome to be shuttled to the host cell. The internal diameter of the tube allows one double-stranded DNA chain to be translocated. We conclude that the assembly principles of the viral tunneling nanotube take advantage of proteo-lipid interactions that confer to the tail tube elastic, mechanical and functional properties employed also in other protein-membrane systems.     

A subset of centrosomal proteins are arranged in a tubular conformation that is reproduced during centrosome duplication

The centrosome plays a fundamental role in organizing the interphase cytoskeleton and the mitotic spindle, and its protein complexity is modulated to support these functions. The centrosome must also duplicate itself once during each cell cycle, thus ensuring the formation of a bipolar spindle and its continuity through successive cell divisions. In this study, we have used a battery of antibodies directed against centrosomal components to study the general organization of the centrosome during the cell cycle and during the centrosome duplication process. We demonstrate that a subset of centrosomal proteins are arranged together to form a tubular pattern within the centrosome. The tubular conformation defined by these proteins has a polarity and is closed at one end. The centriole complement of the centrosome is normally placed near this end. We show that the "wall" of the tube is enriched in proteins such as CDC2, ninein, and pericentrin as well as gamma-tubulin. In addition, a subset of gamma-tubulin is localized to the "lumen" of the tube. We also demonstrate, for the first time, that antibody staining can be used to detect centrosome duplication allowing the identification of duplication intermediates. We show that one product of centrosome duplication is the replication of the tubular structure found within the centrosome. The position of the centriole duplexes prior to and during centrosome duplication is documented and a model of the morphogenesis of the centrosome during the duplication process is proposed.     

Nitrogen removal by tubular gel containing Nitrosomonas europaea and Paracoccus denitrificans.

A new bioreactor for the removal of nitrogen from wastewater is described which consists of a tubular polymeric gel containing Nitrosomonas europaea and Paracoccus denitrificans. The outer surface of the tube is in aerobic contact with wastewater containing ammonia, while the inside of the tube is in anaerobic contact with ethanol flowing through the tube. N. europaea oxidizes ammonia to nitrite in the gel, and then P. denitrificans reduces the nitrite to nitrogen gas in the same gel. This concept would be effective for simplifying nitrogen removal systems requiring aerobic and anaerobic operations.     

A tubular segmented-flow bioreactor for the infection of insect cells with recombinant baculovirus

A continuous process of insect cell (S f9) growth and baculovirus infection is tested with the sequential combination of a CSTR and a tubular reactor. A tubular infection reactor enables continuous introduction of baculovirus and therefore avoids the ‘passage effect’ observed in two-stage CSTR systems. Moreover, a tubular reactor can be used to test cell infection kinetics and the subsequent metabolism of infected insect cells. Unlike batch and CSTR culture, cells in a horizontally positioned tubular reactor settle due to poor mixing. We have overcome this problem by alternately introducing air bubbles and media and by maintaining a linear velocity sufficient to keep cells suspended. This article addresses the development of the tubular reactor and demonstrates its use as an infection system that complements the two-stage CSTR. This revised version was published online in July 2006 with corrections to the Cover Date.     

How to shape a cylinder: pollen tube as a model system for the generation of complex cellular geometry

Expansive growth in plant cells is a formidable problem for biophysical studies, and the mechanical principles governing the generation of complex cellular geometries are still poorly understood. Pollen, the male gametophyte stage of the flowering plants, is an excellent model system for the investigation of the mechanics of complex growth processes. The initiation of pollen tube growth requires first of all, the spatially confined formation of a protuberance. This process must be controlled by the mechanical properties of the cell wall, since turgor is a non-vectorial force. In the elongating tube, cell wall expansion is confined to the apex of the cell, requiring the tubular region to be stabilized against turgor-induced tensile stress. Tip focused surface expansion must be coordinated with the supply of cell wall material to this region requiring the precise, logistical control of intracellular transport processes. The advantage of such a demanding mechanism is the high efficiency it confers on the pollen tube in leading an invasive way of life.     

Nosema notabilis (Microsporidia), its ultrastructure and effect on the myxosporean host Ortholinea polymorpha

Nosema notabilis Kudo, 1939 produces chain-forming meronts with a dense cell coat in direct contact with the host cell cytoplasm. Cytoplasmic microtubules and membranaceous whorls could be observed in meront cytoplasm. Sporonts differ in that they have a thicker cell wall and more conspicuous endoplasmic reticulum (ER) cisternae. Sporoblasts have an externally ridged cell wall. Spores have an apically located anchoring disc, an isofilar polar tube with 6 to 9 turns and polyribosomal strands in the sporoplasm. Diplokarya occur in all stages. Heavily infected plasmodia of Ortholinea polymorpha (Davis, 1917) reveal marked pathological signs. The most prominent are reduction of surface projections and/or pinocytosis, inflated mitochondria with altered inner structures, affected vegetative nuclei, damage to generative cells and occurrence of various anomalous formations in the plasmodium cytoplasm. The damage may result in complete disintegration of the plasmodium. However, the development of the microsporidian is affected by a remarkably high percentage of teratological stages revealing membranaceous and tubular structures.     

A novel fluorescence microscopic approach to quantitatively analyse protein-induced membrane remodelling

Membrane remodelling or the bending and rupture of the lipid bilayer occurs during diverse cellular processes such as cell division, synaptic transmission, vesicular transport, organelle biogenesis and sporulation. These activities are brought about by the localized change in membrane curvature, which in turn causes lipid-packing stress, of a planar lipid bilayer by proteins. For instance, vesicular transport processes are typically characterized by the cooperative recruitment of proteins that induce budding of a planar membrane and catalyse fission of the necks of membrane buds to release vesicles. The analysis of such membrane remodelling reactions has traditionally been restricted to electron microscopy–based approaches or force spectroscopic analysis of membrane tethers pulled from liposome-based model membrane systems. Our recent work has demonstrated the facile creation of tubular model membrane systems of supported membrane tubes (SMrTs), which mimic late-stage intermediates of typical vesicular transport reactions. This review addresses the nature of such an assay system and a fluorescence-intensity-based analysis of changes in tube dimensions that is indicative of the membrane remodelling capacity of proteins.     

THE FISH POND EXPERIMENT: CHAPTER TWO

A tubular photobioreactor for outdoor cultivation of Spirulina platensis was successfully operated for the last two years. The reactor was made of transparent 2.4-cm diameter tubes with a total length of approximately 101-m and a volume of 124-liter. Flow was induced using an airlift pumping system. To optimize the system further, a larger tube diameter was also tested. Preliminary results have suggested that a larger tube diameter might provide increased output with reduced surface area requirements. Results of experiments comparing the productivity of the same culture volume in tubes of 2.4 cm and 5.0 cm will be presented which show a small decrease in productivity by volume, but a large increase in areal productivity. This suggests that the larger tube diameter would be an appropriate choice for larger scale systems. Additionally, data will be presented demonstrating the effectiveness of an on-line surface scatter turbidimeter for accurate measurement of Spirulina density when correlated to manual dry weight measurements.     

The Caenorhabditis elegans sqv genes and functions of proteoglycans in development

In the nematode Caenorhabditis elegans, the vulva is a simple tubular structure linking the gonads with the external cuticle. In this review we summarize knowledge of inter- and intracellular signaling during vulval development and of the genes required for vulval invagination. Mutants of one set of these genes, the sqv genes, have a normal number of vulval precursor cells (VPCs) with an unperturbed cell lineage but the invagination space, normally a tube, is either collapsed or absent. We review evidence that the sqv genes are involved in glycosaminoglycan synthesis and speculate on ways in which defective glycosaminoglycan formation might lead to collapse of the vulval structure.     

藤茶总黄酮对四氯化碳所致大鼠肾损伤保护作用的初步研究

目的：初步探讨藤茶总黄酮（TCF）对四氯化碳所致肾损伤的保护作用。方法：30只雄性SD大鼠被随机分为正常组、模型组和TCF治疗组,第12周处死动物,肾组织石蜡切片,HE染色,光镜下观察肾组织形态结构变化。结果：正常组肾小球及肾小管形态结构无异常;四氯化碳模型组肾的近端小管管腔狭小,上皮细胞萎缩,细胞核浓缩,部分细胞出现空泡变性及少量肾小球毛细血管内皮细胞细胞核出现核浓缩,通透性增加;TCF治疗组肾小球及肾小管形态结构基本恢复正常。结论：藤茶总黄酮对四氯化碳所致大鼠肾损伤有较好的防治作用。     

Cell polarity pathways converge and extend to regulate neural tube closure

Neural tube defects, such as spinabifida, craniorachischisis and anencephaly, are some of the most common birth defects in humans. Recent studies in mouse model systems suggest that craniorachischisis is associated with mutations in genes that regulate cell polarity. Using Xenopus as a model system, Wallingford and Harland have now shed light on the mechanism by which these pathways affect neural tube closure.     

陕西宁强埃迪卡拉系顶部管状化石Sinotubulites形态学与古生态学新知

陕西宁强选将坪地区埃迪卡拉系灯影组高家山段上部的碎屑白云岩中产出大量磷酸盐化三维立体保存的Sinotubulites化石,这为埃迪卡拉纪末期生物的演化及多样性研究提供了一个很好的窗口。文中通过对Si-notubulites的形态结构、纹饰特征、保存特征等方面的观察研究,认为Sinotubulites管体外壁上不规则的褶皱状纹饰是变形过程中展现出的各种形态,而不是管体固有的纹饰形态特征。并且根据管体内外层管壁的不同形态学特征以及在原始生态条件下弯曲生长的管体,将Sinotubulites解释为表栖平躺生物。Sinotubulites可能具有一定的蠕动能力,而不规则褶皱状外壁在变形过程中可以产生微弱的蠕动力,使得管体对自身的位置得以调整。     

KAP degradation by calpain is associated with CK2 phosphorylation and provides a novel mechanism for cyclosporine A-induced proximal tubule injury

The use of cyclosporine A (CsA) is limited by its severe nephrotoxicity that includes reversible vasoconstrictor effects and proximal tubule cell injury, the latter associated whith chronic kidney disease progression. The mechanisms of CsA-induced tubular injury, mainly on the S3 segment, have not been completely elucidated. Kidney androgen-regulated protein (KAP) is exclusively expressed in kidney proximal tubule cells, interacts with the CsA-binding protein cyclophilin B and its expression diminishes in kidneys of CsA-treated mice. Since we reported that KAP protects against CsA toxicity in cultured proximal tubule cells, we hypothesized that low KAP levels found in kidneys of CsA-treated mice might correlate with proximal tubule cell injury. To test this hypothesis, we used KAP Tg mice developed in our laboratory and showed that these mice are more resistant to CsA-induced tubular injury than control littermates. Furthermore, we found that calpain, which was activated by CsA in cell cultures and kidney, is involved in KAP degradation and observed that phosphorylation of serine and threonine residues found in KAP PEST sequences by protein kinase CK2 enhances KAP degradation by calpain. Moreover, we also observed that CK2 inhibition protected against CsA-induced cytotoxicity. These findings point to a novel mechanism for CsA-induced kidney toxicity that might be useful in developing therapeutic strategies aimed at preventing tubular cell damage while maintaining the immunosuppressive effects of CsA.