scholarly journals Traffic of Chitin Synthase 1 (CHS-1) to the Spitzenkörper and Developing Septa in Hyphae of Neurospora crassa: Actin Dependence and Evidence of Distinct Microvesicle Populations

2011 ◽  
Vol 10 (5) ◽  
pp. 683-695 ◽  
Author(s):  
Eddy Sánchez-León ◽  
Jorge Verdín ◽  
Michael Freitag ◽  
Robert W. Roberson ◽  
Salomon Bartnicki-Garcia ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Neurospora Crassa ◽  
Laser Scanning ◽  
Fine Particles ◽  
Fluorescent Protein ◽  
Apical Cell ◽  
Subcellular Location ◽  
Chitin Synthase ◽  
Wide Field ◽  
Chitin Synthases

ABSTRACTWe describe the subcellular location of chitin synthase 1 (CHS-1), one of seven chitin synthases inNeurospora crassa. Laser scanning confocal microscopy of growing hyphae showed CHS-1–green fluorescent protein (GFP) localized conspicuously in regions of active wall synthesis, namely, the core of the Spitzenkörper (Spk), the apical cell surface, and developing septa. It was also present in numerous fine particles throughout the cytoplasm plus some large vacuoles in distal hyphal regions. Although the same general subcellular distribution was observed previously for CHS-3 and CHS-6, they did not fully colocalize. Dual labeling showed that the three different chitin synthases were contained in different vesicular compartments, suggesting the existence of a different subpopulation of chitosomes for each CHS. CHS-1–GFP persisted in the Spk during hyphal elongation but disappeared from the septum after its development was completed. Wide-field fluorescence microscopy and total internal reflection fluorescence microscopy revealed subapical clouds of particles, suggestive of chitosomes moving continuously toward the Spk. Benomyl had no effect on CHS-1–GFP localization, indicating that microtubules are not strictly required for CHS trafficking to the hyphal apex. Conversely, actin inhibitors caused severe mislocalization of CHS-1–GFP, indicating that actin plays a major role in the orderly traffic and localization of CHS-1 at the apex.

scholarly journals Spitzenkörper Localization and Intracellular Traffic of Green Fluorescent Protein-Labeled CHS-3 and CHS-6 Chitin Synthases in Living Hyphae of Neurospora crassa

2007 ◽  
Vol 6 (10) ◽  
pp. 1853-1864 ◽  
Author(s):  
Meritxell Riquelme ◽  
Salomon Bartnicki-García ◽  
Juan Manuel González-Prieto ◽  
Eddy Sánchez-León ◽  
Jorge A. Verdín-Ramos ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Neurospora Crassa ◽  
Laser Scanning ◽  
Fluorescent Dyes ◽  
Fluorescent Protein ◽  
Golgi Body ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Chitin Synthases ◽  
Green Fluorescent

ABSTRACT The subcellular location and traffic of two selected chitin synthases (CHS) from Neurospora crassa, CHS-3 and CHS-6, labeled with green fluorescent protein (GFP), were studied by high-resolution confocal laser scanning microscopy. While we found some differences in the overall distribution patterns and appearances of CHS-3-GFP and CHS-6-GFP, most features were similar and were observed consistently. At the hyphal apex, fluorescence congregated into a conspicuous single body corresponding to the location of the Spitzenkörper (Spk). In distal regions (beyond 40 μm from the apex), CHS-GFP revealed a network of large endomembranous compartments that was predominantly comprised of irregular tubular shapes, while some compartments were distinctly spherical. In the distal subapex (20 to 40 μm from the apex), fluorescence was observed in globular bodies that appeared to disintegrate into vesicles as they advanced forward until reaching the proximal subapex (5 to 20 μm from the apex). CHS-GFP was also conspicuously found delineating developing septa. Analysis of fluorescence recovery after photobleaching suggested that the fluorescence of the Spk originated from the advancing population of microvesicles (chitosomes) in the subapex. The inability of brefeldin A to interfere with the traffic of CHS-containing microvesicles and the lack of colocalization of CHS-GFP with the endoplasmic reticulum (ER)-Golgi body fluorescent dyes lend support to the idea that CHS proteins are delivered to the cell surface via an alternative route distinct from the classical ER-Golgi body secretory pathway.

scholarly journals Green Fluorescent Chimeras Indicate Nonpolar Localization of Pullulanase Secreton Components PulL and PulM

2006 ◽  
Vol 188 (8) ◽  
pp. 2928-2935 ◽  
Author(s):  
Nienke Buddelmeijer ◽  
Olivera Francetic ◽  
Anthony P. Pugsley
Keyword(s):  
Fluorescence Microscopy ◽  
Fluorescent Protein ◽  
Cell Envelope ◽  
Klebsiella Oxytoca ◽  
Subcellular Location ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Native Proteins ◽  
Type Ii Secretion System ◽  
Green Fluorescent

ABSTRACT The Klebsiella oxytoca pullulanase secreton (type II secretion system) components PulM and PulL were tagged at their N termini with green fluorescent protein (GFP), and their subcellular location was examined by fluorescence microscopy and fractionation. When produced at moderate levels without other secreton components in Escherichia coli, both chimeras were envelope associated, as are the native proteins. Fluorescent GFP-PulM was evenly distributed over the cell envelope, with occasional brighter foci. Under the same conditions, GFP-PulL was barely detectable in the envelope by fluorescence microscopy. When produced together with all other secreton components, GFP-PulL exhibited circumferential fluorescence, with numerous brighter patches. The envelope-associated fluorescence of GFP-PulL was almost completely abolished when native PulL was also produced, suggesting that the chimera cannot compete with PulL for association with other secreton components. The patches of GFP-PulL might represent functional secretons, since GFP-PulM also appeared in similar patches. GFP-PulM and GFP-PulL both appeared in spherical polar foci when made at high levels. In K. oxytoca, GFP-PulM was evenly distributed over the cell envelope, with few patches, whereas GFP-PulL showed only weak envelope-associated fluorescence. These data suggest that, in contrast to their Vibrio cholerae Eps secreton counterparts (M. Scott, Z. Dossani, and M. Sandkvist, Proc. Natl. Acad. Sci. USA 98:13978-13983, 2001), PulM and PulL do not localize specifically to the cell poles and that the Pul secreton is distributed over the cell surface.

scholarly journals The Plasma Membrane Proton Pump PMA-1 Is Incorporated into Distal Parts of the Hyphae Independently of the Spitzenkörper in Neurospora crassa

2013 ◽  
Vol 12 (8) ◽  
pp. 1097-1105 ◽  
Author(s):  
Rosa A. Fajardo-Somera ◽  
Barry Bowman ◽  
Meritxell Riquelme
Keyword(s):  
Plasma Membrane ◽  
Neurospora Crassa ◽  
Nutrient Uptake ◽  
Laser Scanning ◽  
Germ Tube ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Fungal Growth ◽  
Electrochemical Gradient ◽  
Content Type

ABSTRACT Most models for fungal growth have proposed a directional traffic of secretory vesicles to the hyphal apex, where they temporarily aggregate at the Spitzenkörper before they fuse with the plasma membrane (PM). The PM H + -translocating ATPase (PMA-1) is delivered via the classical secretory pathway (endoplasmic reticulum [ER] to Golgi) to the cell surface, where it pumps H + out of the cell, generating a large electrochemical gradient that supplies energy to H + -coupled nutrient uptake systems. To characterize the traffic and delivery of PMA-1 during hyphal elongation, we have analyzed by laser scanning confocal microscopy (LSCM) strains of Neurospora crassa expressing green fluorescent protein (GFP)-tagged versions of the protein. In conidia, PMA-1-GFP was evenly distributed at the PM. During germination and germ tube elongation, PMA-1-GFP was found all around the conidial PM and extended to the germ tube PM, but fluorescence was less intense or almost absent at the tip. Together, the data indicate that the electrochemical gradient driving apical nutrient uptake is generated from early developmental stages. In mature hyphae, PMA-1-GFP localized at the PM at distal regions (>120 μm) and in completely developed septa, but not at the tip, indicative of a distinct secretory route independent of the Spitzenkörper occurring behind the apex.

scholarly journals Estimating numbers of fluorescent molecules in single cells by analysing fluctuations in photobleaching

2018 ◽  
Author(s):  
Elco Bakker ◽  
Peter S. Swain
Keyword(s):  
Fluorescence Microscopy ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Single Cells ◽  
Measurement Noise ◽  
Wide Field ◽  
Stochastic Fluctuations ◽  
Biochemical Measurements ◽  
Green Fluorescent ◽  
The Impact

The impact of fluorescence microscopy has been limited by the difficulties of express-ing measurements of fluorescent proteins in numbers of molecules. Absolute numbers enable the integration of results from different laboratories, empower mathematical modelling, and are the bedrock for a quantitative, predictive biology. Here we develop a general algorithm to infer numbers of molecules from fluctuations in the photobleaching of proteins tagged with Green Fluorescent Protein. To untangle measurement noise from stochastic fluctuations, we use the linear noise approximation and Kalman filtering within a framework of Bayesian inference. Not only do our results agree with biochemical measurements for multiple proteins in budding yeast, but we also provide a statistically verified model of measurement noise for fluorescence microscopes. The experiments we require are straightforward and use only a wide-field fluorescence microscope. As such, our approach has the potential to become standard for those practising quantitative fluorescence microscopy.

scholarly journals Estimating numbers of intracellular molecules through analysing fluctuations in photobleaching

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Elco Bakker ◽  
Peter S. Swain
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescence Microscopy ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Wide Field ◽  
Order Of Magnitude ◽  
Biochemical Measurements ◽  
Green Fluorescent ◽  
The Impact ◽  
Quantitative Fluorescence

Abstract The impact of fluorescence microscopy has been limited by the difficulties of expressing measurements of fluorescent proteins in numbers of molecules. Absolute numbers enable the integration of results from different laboratories, empower mathematical modelling, and are the bedrock for a quantitative, predictive biology. Here we propose an estimator to infer numbers of molecules from fluctuations in the photobleaching of proteins tagged with Green Fluorescent Protein. Performing experiments in budding yeast, we show that our estimates of numbers agree, within an order of magnitude, with published biochemical measurements, for all six proteins tested. The experiments we require are straightforward and use only a wide-field fluorescence microscope. As such, our approach has the potential to become standard for those practising quantitative fluorescence microscopy.

scholarly journals High Resolution 3-D Fluorescence Microscopy: A Comparison of Confocal Laser Scanning Microscopy and a wide-Field Deconvolution Technique

1997 ◽  
Vol 5 (6) ◽  
pp. 10-13 ◽  
Author(s):  
Keyword(s):  
Biological Sciences ◽  
Fluorescence Microscopy ◽  
Light Microscopy ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Specific Structure ◽  
Confocal Laser ◽  
Wide Field ◽  
Deconvolution Technique ◽  
Transmitted Light Microscopy

Advances in the biological sciences have given rise for the need to visualize microscopic structures of interest. As the requirement to see specific structure and sub-resolution details arise, investigators have turned to fluorescent probes to label and observe these details, which are often undetectable using conventional methods in light microscopy. Fluorescence microscopy offers many advantages for visualizing specific structure and sub-resolution details, which are often undetectable using transmitted light microscopy. Recent advances in fluorescent probe and microscope design, as well as imaging instrumentation designed for fluorescence applications, are now permitting life-science researchers to view details in regions of interest with increasing precision, accuracy, and resolution.

scholarly journals Class III Chitin Synthase ChsB of Aspergillus nidulans Localizes at the Sites of Polarized Cell Wall Synthesis and Is Required for Conidial Development

2009 ◽  
Vol 8 (7) ◽  
pp. 945-956 ◽  
Author(s):  
Kazuharu Fukuda ◽  
Kazunari Yamada ◽  
Ken Deoka ◽  
Shuichi Yamashita ◽  
Akinori Ohta ◽  
...  
Keyword(s):  
Aspergillus Nidulans ◽  
Cell Walls ◽  
Deletion Mutant ◽  
Fluorescent Protein ◽  
Hyphal Growth ◽  
Chitin Synthase ◽  
Class Iii ◽  
Chitin Synthases ◽  
Conidial Development ◽  
Green Fluorescent

ABSTRACT Class III chitin synthases play important roles in tip growth and conidiation in many filamentous fungi. However, little is known about their functions in those processes. To address these issues, we characterized the deletion mutant of a class III chitin synthase-encoding gene of Aspergillus nidulans, chsB, and investigated ChsB localization in the hyphae and conidiophores. Multilayered cell walls and intrahyphal hyphae were observed in the hyphae of the chsB deletion mutant, and wavy septa were also occasionally observed. ChsB tagged with FLAG or enhanced green fluorescent protein (EGFP) localized mainly at the tips of germ tubes, hyphal tips, and forming septa during hyphal growth. EGFP-ChsB predominantly localized at polarized growth sites and between vesicles and metulae, between metulae and phialides, and between phalides and conidia in asexual development. These results strongly suggest that ChsB functions in the formation of normal cell walls of hyphae, as well as in conidiophore and conidia development in A. nidulans.

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