Microtubules and angiosperm bordered pit formation

Summary Microtubules have been shown to run around the perimeter of the pit aperture of developing bordered pits of Salix fragilis, L. These microtubules are parallel to the microfibrils being produced and do not converge with them. Although microtubules may be parallel to microfibrils in differentiating vessel elements as well as in fibres, many cases have been found where microtubules are not so orientated. There is no direct evidence for the involvement of microtubules in the orientation of microfibrils in the secondary wall, although their position during wall synthesis suggests some ancillary role.     

Cargo regulates clathrin-coated pit dynamics

Clathrin-coated pits (CCPs) are generally considered a uniform population of endocytic machines containing mixed constitutive and regulated membrane cargo. Contrary to this view, we show that regulated endocytosis of G protein-coupled receptors (GPCRs) occurs preferentially through a subset of CCPs. Significantly, GPCR-containing CCPs are also functionally distinct, as their surface residence time is regulated locally by GPCR cargo via PDZ-dependent linkage to the actin cytoskeleton. Such cargo-regulated CCPs show delayed recruitment of dynamin and can undergo an abortive event in which clathrin coats separate from the plasma membrane without concomitant receptor endocytosis. Segregation of cargo into CCP subsets, combined with cargo-dependent control of CCP dynamics, suggests a simple kinetic mechanism to generate functional specialization early in the endocytic pathway and reduce competition between diverse endocytic cargo.     

Synaptotagmin regulation of coated pit assembly

Synaptotagmins bind clathrin AP-2 with high affinity via their second C(2) domain, which indicates they are involved in coated pit function. We now report that expression of synaptotagmins lacking either the second C(2) domain or the entire cytoplasmic region potently inhibit endocytosis. Inhibition was dependent on two intramembrane cysteine residues that were found to be essential for synaptotagmin oligomerization. Cells expressing the wild-type, but not the mutant, truncated synaptotagmin fragment had a reduced number of clathrin-coated pits. These results suggest that the formation of synaptotagmin multimers is an important step in the regulation of coated pit assembly.     

Stochastic model of clathrin-coated pit assembly

In recent years, fluorescence microscopy has enabled researchers to observe the dynamics of clathrin-coated pit (CCP) assembly in real time. The assembly dynamics of CCPs shows striking heterogeneity. Some CCPs are long-lived (productive CCPs); they bind cargo and grow in size to form clathrin-coated vesicles. In contrast, other CCPs (abortive CCPs) are relatively short-lived and disassemble well before reaching vesicle size. Within both populations there is significant variance in CCP lifetime. We propose a stochastic biophysical model that links these observations with the energetics of CCPs and kinetics of their assembly. We show that without cargo, CCP assembly faces a high energy barrier that is difficult to overcome. As a consequence, CCPs without cargo are almost always abortive. We suggest a mechanism by which cargo binding stabilizes CCPs and facilitates their growth. The lifetime distribution of abortive pits calculated from our model agrees well with published experimental data. We also estimate the lifetimes of productive CCPs and show that the stochastic nature of CCP assembly plays a crucial role in causing their observed wide distribution.     

Analysis of circular bordered pit function II. Gymnosperm tracheids with torus-margo pit membranes

A model of xylem conduit function was applied to gymnosperm tracheids with torus-margo pit membranes for comparison with angiosperm vessels. Tracheids from 17 gymnosperm tree species with circular bordered pits and air-seed pressures from 0.8 to 11.8 MPa were analyzed. Tracheids were more reinforced against implosion than vessels, consistent with their double function in transport and support. Tracheid pits were 3.3 to 44 times higher in hydraulic conductivity than vessel pits because of greater membrane conductivity of the torus-margo configuration. Tight scaling between torus and pit size maximized pit conductivity. Higher pit conductivity allowed tracheids to be 1.7-3.4 times shorter than vessels and still achieve 95% of their lumen-limited maximum conductivity. Predicted tracheid lengths were consistent with measured lengths. The torus-margo structure is important for maximizing the conductivity of the inherently length-limited tracheid: replacing the torus-margo membrane with a vessel membrane caused stem tracheid conductivity to drop by 41%. Tracheids were no less hydraulically efficient than vessels if they were long enough to reach their lumen-limiting conductivity. However, this may only be possible for lumen diameters below approximately 60-70 μm.     

Fishing for clathrin-coated pit nucleators

The membrane-curvature-inducing protein Fcho was proposed to be part of a ubiquitous nucleation mechanism for clathrin-coated pits. However, studies in developing zebrafish embryos now indicate a role for Fcho as a receptor-specific adaptor in bone morphogenetic protein (BMP) signalling, rather than a global coated-pit nucleator.     

厌氧产酸菌群培养技术在窖泥质量快速检测中的应用

【背景】窖泥质量决定了浓香型白酒品质的高低,窖泥中的产酸功能菌群显著影响着窖泥的质量及对应的浓香型白酒品质,但目前对窖泥质量的评价尚缺乏明确和完善的标准。【目的】建立一种快速、准确评价窖泥质量的方法,解析窖泥中参与己酸合成的重要微生物。【方法】通过窖泥产酸菌群培养和产酸代谢特征研究,构建厌氧产酸菌群发酵体系,根据窖泥中厌氧菌的己酸合成能力来评价相应窖泥的质量;利用高通量测序技术分析可培养发酵体系中产己酸功能微生物的群落组成。【结果】葡萄糖碳源相比乳酸碳源可以更有效地富集窖泥中的产己酸菌群;采用毫升级别发酵体系、对窖底泥进行多点取样,产酸菌群发酵代谢产物稳定期的己酸产量、己酸/丁酸值可较准确地评价不同窖泥的质量。16S rRNA基因测序结果表明,利用产酸菌群培养方法可以有效地富集未培养产己酸细菌(Uncultured bacterium Caproiciproducens)。【结论】基于产酸菌群培养的窖泥微生物发酵体系可用于实际生产中快速、准确地评价窖泥质量,并为窖泥微生物己酸合成代谢机制的研究提供参考。     

Analysis of circular bordered pit function I. Angiosperm vessels with homogenous pit membranes

A model predicted pit and vessel conductivity, the air-seed pressure for cavitation, and the implosion pressure causing vessel collapse. Predictions were based on measurements from 27 angiosperm species with circular bordered pits and air-seed pressures of 0.2-11.3 MPa. Vessel implosion pressure exceeded air-seed pressure by a safety factor of 1.8 achieved by the increase in vessel wall thickness per vessel diameter with air-seed pressure. Intervessel pitting reduced the implosion pressure by 20 to 40%. Pit hydraulic conductivity decreased by 30-fold from low (<1 MPa) to high (>10 MPa) air-seed pressure primarily because of decreasing pit membrane conductivity. Vessel conductivity (per length and wall area) increased with vessel length as higher lumen conductivity overcame low pit conductivity. At the "saturating vessel length," vessel conductivity maximized at the Hagen-Poiseuille value for the lumen per wall area. Saturated vessel conductivity declined by sixfold with increasing air-seed pressure because of increased wall thickness associated with increased implosion resistance. The saturated vessel length is likely the optimal length because: (a) shorter vessels have lower conductivities, (b) longer vessels do not increase conductivity when functional yet decrease it more when cavitated, (c) observed pit structure most closely optimized vessel conductivity at the saturated length, and (d) saturated lengths were similar to measured lengths.     

Analysis of coated pit recycling on human fibroblasts

The recycling to the cell surface of previously internalized coated pits has been proposed as a likely mechanism for the rapid regeneration of coated pits on human fibroblast surfaces at 37 degrees C (1). We present a general mathematical model of coated pit recycling for the case when the coat cycles as a single unit, and use it to analyze certain time and temperature dependent data obtained by Anderson et al. (1) and Vermeer et al. (2). We show how recycling can account for these data and how this type of data can be used to distinguish between different possible recycling mechanisms. We show that these data are inconsistent with a two compartment model where coat material simply shuttles back and forth between coated pits and short-lived coated vesicles. From these data we estimate for human fibroblasts at 37 degrees C: that the time for a coated pit to be replenished through recycling after it is lost through internalization is greater than 3.5 min; and that at any moment 53% or less of the cell's clathrin that is involved in coated pit recycling is on the cell surface.     

Analysis of coated pit recycling on human fibroblasts

The recycling to the cell surface of previously internalized coated pits has been proposed as a likely mechanism for the rapid regeneration of coated pits on human fibroblast surfaces at 37°C (1). We present a general mathematical model of coated pit recycling for the case when the coat cycles as a single unit, and use it to analyze certain time and temperature dependent data obtained by Anderson et al. (1) and Vermeer et al. (2). We show how recycling can account for these data and how this type of data can be used to distinguish between different possible recycling mechanisms. We show that these data are inconsistent with a two compartment model where coat material simply shuttles back and forth between coated pits and short-lived coated vesicles. From these data we estimate for human fibroblasts at 37°C: that the time for a coated pit to be replenished through recycling after it is lost through internalization is greater than 3.5 min; and that at any moment 53% or less of the cell’s clathrin that is involved in coated pit recycling is on the cell surface.     

Variation in pit size of antlion (Myrmeleon carolinus) larvae: the importance of pit construction

Abstract.Larvae of several antlions build pits that vary in size across and within species. The influence of food limitation and pit building experience on variation in pit size of the larvae of Myrmeleon carolinus was investigated in the laboratory. Unfed larvae that were allowed to build pits had smaller pit diameters than fed larvae. However, fed antlions that had been previously prevented from pit building, initially did not build larger pits than unfed antlion larvae that, too, had been prevented from pit building. Therefore, physiological constraints associated with food limitation alone are not sufficient to explain the reduction in pit size of food limited antlions of this species.     

Plasmodesmata and pit development in secondary xylem elements

Developing pit membranes of secondary xylem elements in Drimys winteri, Fagus sylvatica, Quercus robur, Sorbus aucuparia, Tilia vulgaris and Trochodendron aralioides have been examined by transmission electron microscopy. Absence of plasmodesmata from the membranes of vessel elements and tracheids indicates that their pits develop independently of these structures. On the other hand, plasmodesmata are abundant in pit membranes between fibres, parenchyma cells, and combinations of these cell types in Fagus, Quercus and Tilia. In each case the plasmodesmata pass right through the developing pit membrane. In the case of Sorbus fibres, however, plasmodesmata were absent from the majority of pit membrane profiles seen in sections. Occasionally they were observed in large numbers associated with a swollen region on one side of the pit membrane between fibres and between fibres and parenchyma, radiating from a small area of the middle lamella. In the case of fibre to parenchyma pitting, this swelling was always found on the fibre side of the membrane, while on the other side a small number of plasmodesmata were present completing communication with the parenchyma cytoplasm. These observations are discussed with regard to the role of plasmodesmata in pit formation, and in the differentiation of the various cell types in secondary xylem. The significance their distribution may have for our understanding of xylem evolution is also discussed.     

Substrate particle size affects pit building decision and pit size in the antlion larvae Euroleon nostras (Neuroptera: Myrmeleontidae)

Abstract. The larvae of the antlion Euroleon nostras are pit-builders, constructing pitfall traps in loose sand. The number of pits and the pit diameter are recorded when larvae are kept in substrates with different particle sizes. The most convenient pit-building sand fractions are two fractions with fine sand (≤ 0.23 mm; 0.23–0.54 mm). The largest pits are constructed in sand with a particle size of 0.23–0.54 mm. In this sand fraction, larvae of all three instars most readily build pits. No pits are constructed in sand with a particle size greater than 1.54 mm. First- and second-instar larvae avoid building pits in substrates of particle size 1–1.54 mm, but third-instar larvae construct pits in this sand fraction. It is assumed that the antlion is capable of distinguishing between substrate types and this hypothesis is tested by giving larvae the choice of building a pit in one of four particle-size fractions. Larvae of all three instars prefer to build pits in the fraction with a particle size of 0.23–0.54 mm. Only third-instar larvae build pits in all four fractions, but only occasionally in the coarser fraction.     

The pit organs of elasmobranchs: a review

Elasmobranchs have hundreds of tiny sensory organs, called pit organs, scattered over the skin surface. The pit organs were noted in many early studies of the lateral line, but their exact nature has long remained a mystery. Although pit organs were known to be innervated by the lateral line nerves, and light micrographs suggested that they were free neuromasts, speculation that they may be external taste buds or chemoreceptors has persisted until recently. Electron micrographs have now revealed that the pit organs are indeed free neuromasts. Their functional and behavioural role(s), however, are yet to be investigated.     

The comparative morphology of pit organs in elasmobranchs

The pit organs of elasmobranchs (sharks, skates and rays) are free neuromasts of the mechanosensory lateral line system. Pit organs, however, appear to have some structural differences from the free neuromasts of bony fishes and amphibians. In this study, the morphology of pit organs was investigated by scanning electron microscopy in six shark and three ray species. In each species, pit organs contained typical lateral line hair cells with apical stereovilli of different lengths arranged in an “organ‐pipe” configuration. Supporting cells also bore numerous apical microvilli taller than those observed in other vertebrate lateral line organs. Pit organs were either covered by overlapping denticles, located in open grooves bordered by denticles, or in grooves without associated denticles. The possible functional implications of these morphological features, including modification of water flow and sensory filtering properties, are discussed. J. Morphol. 2009. © 2009 Wiley‐Liss, Inc.     

Python pit organs analyzed as warm receptors

The infrared receptor neurons of Python reticulatuspit organs were all found to have bimodal sensitivity, responding to both infrared and touch stimuli with fairly rapid adaptation. The majority (22 of 29 neurons) had no background discharges at any temperature between 20 and 33°C. The receptive areas were 150–250 µm in diameter and identical for both modalities. There was only one receptive area for each neuron. These facts suggest the possibility that some kinds of temperature sensitive neurons can also function as touch neurons and vice versa, not only in this species, but also in other animals.     

Local clustering of transferrin receptors promotes clathrin-coated pit initiation

Clathrin-mediated endocytosis (CME) is the major pathway for concentrative uptake of receptors and receptor-ligand complexes (cargo). Although constitutively internalized cargos are known to accumulate into maturing clathrin-coated pits (CCPs), whether and how cargo recruitment affects the initiation and maturation of CCPs is not fully understood. Previous studies have addressed these issues by analyzing the global effects of receptor overexpression on CME or CCP dynamics. Here, we exploit a refined approach using expression of a biotinylated transferrin receptor (bTfnR) and controlling its local clustering using mono- or multivalent streptavidin. We show that local clustering of bTfnR increased CCP initiation. By tracking cargo loading in individual CCPs, we found that bTfnR clustering preceded clathrin assembly and confirmed that bTfnR-containing CCPs mature more efficiently than bTfnR-free CCPs. Although neither the clustering nor the related changes in cargo loading altered the rate of CCP maturation, bTfnR-containing CCPs exhibited significantly longer lifetimes than other CCPs within the same cell. Together these results demonstrate that cargo composition is a key source of the differential dynamics of CCPs.