Quantitative single molecule RNA-FISH and RNase-free cell wall digestion in Neurospora crassa

Single molecule RNA-FISH (smFISH) is a valuable tool for analysis of mRNA spatial patterning in fixed cells that is underutilized in filamentous fungi. A primary complication for fixed-cell imaging in filamentous fungi is the need for enzymatic cell wall permeabilization, which is compounded by considerable variability in cell wall composition between species. smFISH adds another layer of complexity due to a requirement for RNase free conditions. Here, we describe the cloning, expression, and purification of a chitinase suitable for supplementation of a commercially available RNase-free enzyme preparation for efficient permeabilization of the Neurospora cell wall. We further provide a method for smFISH in Neurospora which includes a tool for generating numerical data from images that can be used in downstream customized analysis protocols.

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Ultrastructure and Morphology of the Neurospora Crassa Cell Wall Mutants, Slime And Slime X, Using Tem and Sem

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100090257 ◽

1976 ◽

Vol 34 ◽

pp. 54-55

Author(s):

Karen S. Howard ◽

H. D. Braymer ◽

M. D. Socolofsky ◽

S. A. Milligan

Keyword(s):

Cell Wall ◽

Neurospora Crassa ◽

Wild Type ◽

Morphological Comparison ◽

Small Areas ◽

Solid Media ◽

Thin Sectioning ◽

X Cells ◽

Mutant Cells ◽

Isolated Cell

The recently isolated cell wall mutant slime X of Neurospora crassa was prepared for ultrastructural and morphological comparison with the cell wall mutant slime. The purpose of this article is to discuss the methods of preparation for TEM and SEM observations, as well as to make a preliminary comparison of the two mutants.TEM: Cells of the slime mutant were prepared for thin sectioning by the method of Bigger, et al. Slime X cells were prepared in the same manner with the following two exceptions: the cells were embedded in 3% agar prior to fixation and the buffered solutions contained 5% sucrose throughout the procedure.SEM: Two methods were used to prepare mutant and wild type Neurospora for the SEM. First, single colonies of mutant cells and small areas of wild type hyphae were cut from solid media and fixed with OSO4 vapors similar to the procedure used by Harris, et al. with one alteration. The cell-containing agar blocks were dehydrated by immersion in 2,2-dimethoxypropane (DMP).

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Treadmilling FtsZ polymers drive the directional movement of sPG-synthesis enzymes via a Brownian ratchet mechanism

Nature Communications ◽

10.1038/s41467-020-20873-y ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Joshua W. McCausland ◽

Xinxing Yang ◽

Georgia R. Squyres ◽

Zhixin Lyu ◽

Kevin E. Bruce ◽

...

Keyword(s):

Cell Wall ◽

Single Molecule ◽

Theoretical Modelling ◽

Binding Potential ◽

Central Component ◽

Macromolecular Complex ◽

Bacterial Cells ◽

Brownian Ratchet ◽

Ratchet Mechanism ◽

Directional Movement

AbstractThe FtsZ protein is a central component of the bacterial cell division machinery. It polymerizes at mid-cell and recruits more than 30 proteins to assemble into a macromolecular complex to direct cell wall constriction. FtsZ polymers exhibit treadmilling dynamics, driving the processive movement of enzymes that synthesize septal peptidoglycan (sPG). Here, we combine theoretical modelling with single-molecule imaging of live bacterial cells to show that FtsZ’s treadmilling drives the directional movement of sPG enzymes via a Brownian ratchet mechanism. The processivity of the directional movement depends on the binding potential between FtsZ and the sPG enzyme, and on a balance between the enzyme’s diffusion and FtsZ’s treadmilling speed. We propose that this interplay may provide a mechanism to control the spatiotemporal distribution of active sPG enzymes, explaining the distinct roles of FtsZ treadmilling in modulating cell wall constriction rate observed in different bacteria.

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Two cell wall .BETA.-D-glucans from Neurospora crassa.

Agricultural and Biological Chemistry ◽

10.1271/bbb1961.47.1317 ◽

1983 ◽

Vol 47 (6) ◽

pp. 1317-1322 ◽

Cited By ~ 8

Author(s):

Nozomi HIURA ◽

Tasuku NAKAJIMA ◽

Kazuo MATSUDA

Keyword(s):

Cell Wall ◽

Neurospora Crassa

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Single Molecule RNA FISH (smFISH) in Whole‐Mount Mouse Embryonic Organs

Current Protocols in Cell Biology ◽

10.1002/cpcb.79 ◽

2018 ◽

Vol 83 (1) ◽

Author(s):

Shaohe Wang

Keyword(s):

Single Molecule ◽

Rna Fish ◽

Whole Mount

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Transcription factor residence time dominates over concentration in transcription activation

10.1101/2020.11.26.400069 ◽

2020 ◽

Author(s):

Achim P. Popp ◽

Johannes Hettich ◽

J. Christof M. Gebhardt

Keyword(s):

Transcription Factor ◽

Residence Time ◽

Single Molecule ◽

Search Time ◽

Mrna Synthesis ◽

Burst Frequency ◽

Quantitative Measurements ◽

Rna Fish ◽

Transition Times ◽

Basic Course

Transcription is a vital process activated by transcription factor (TF) binding. The active gene releases a burst of transcripts before turning inactive again. While the basic course of transcription is well understood, it is unclear how binding of a TF affects the frequency, duration and size of a transcriptional burst. We systematically varied the residence time and concentration of a synthetic TF and characterized the transcription of a reporter gene by combining single molecule imaging, single molecule RNA-FISH, live transcript visualisation and analysis with a novel algorithm, Burst Inference from mRNA Distributions (BIRD). For this well-defined system, we found that TF binding solely affected burst frequency and variations in TF residence time had a stronger influence than variations in concentration. This enabled us to device a model of gene transcription, in which TF binding triggers multiple successive steps before the gene transits to the active state and actual mRNA synthesis is decoupled from TF presence. We quantified all transition times of the TF and the gene, including the TF search time and the delay between TF binding and the onset of transcription. Our quantitative measurements and analysis revealed detailed kinetic insight, which may serve as basis for a bottom-up understanding of gene regulation.

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