3-d-time-lapse-imaging-cell-wall-dynamics-using-calcofluor-moss

3-D Time-Lapse Imaging of Cell Wall Dynamics Using Calcofluor in the Moss <em>Physcomitrium patens</em>

Time-lapse imaging with fluorescence microscopy allows observation of the dynamic changes of growth and development at cellular and subcellular levels. In ...

Neuromyelitis optica (NMO) is associated with antibodies to aquaporin 4 (AQP4). We hypothesized that antibodies to AQP4 can be triggered by exposure to environmental proteins. We compared human AQP4 to plant and bacterial proteins to investigate the occurrence of significantly similar structures and sequences. High similarity to a known epitope for NMO-IgG, AQP4(207-232), was observed for corn ZmTIP4-1. NMO and non-NMO sera were assessed for reactivity to AQP4(207-232) and the corn peptide. NMO patient serum showed reactivity to both peptides as well as to plant tissue. These findings warrant further investigation into the role of the environment in NMO etiology.

Aquaporin 4 molecular mimicry and implications for neuromyelitis optica.

Most eukaryotic proteins are post-translationally modified, and modification has profound effects on protein function. One key modification is the attachment of a lipid group to certain amino acids; this typically facilitates subcellular targeting (association with a membrane) and protein-protein interactions (by virtue of the large hydrophobic moiety). Most widely recognized are lipid modifications of proteins involved in developmental signaling, but proteins with structural roles are also lipid-modified. The three known types of intracellular protein lipid modifications are S-acylation, N-myristoylation, and prenylation. In plants, genetic analysis of the enzymes involved, along with molecular analysis of select target proteins, has recently shed light on the roles of lipid modification in key developmental processes, such as meristem function, flower development, polar cell elongation, cell differentiation, and hormone responses. In addition, while lipid post-translational mechanisms are generally conserved among eukaryotes, plants differ in the nature and function of target proteins, the effects of lipid modification on target proteins, and the roles of lipid modification in developmental processes.

The role of lipid post-translational modification in plant developmental processes.

Prenylation is a series of lipid posttranslational modifications that are involved in several key aspects of plant development. We recently knocked out every prenylation subunit in Physcomitrella patens. Like in Arabidopsis, knockout of protein farnesyltransferase and protein geranylgeranyltransferase in P. patens does not result in lethality; however, effects on development are extensive. In particular, the knockout of protein geranylgeranyltransferase results in small unicellular plants that resemble algae. Here we perform an analysis of predicted geranylgeranyltransferase target proteins in P. patens, and draw attention to those most likely to play a role in the knockout phenotype.

Turning moss into algae: prenylation targets in Physcomitrella patens.

The circularly permuted GTPase large subunit GTPase 1 (LSG1) is involved in the maturation step of the 60S ribosome and is essential for cell viability in yeast. Here, an Arabidopsis mutant dig6 (drought inhibited growth of lateral roots) was isolated. The mutant exhibited multiple auxin-related phenotypes, which included reduced lateral root number, altered leaf veins, and shorter roots. Genetic mapping combined with next-generation DNA sequencing identified that the mutation occurred in AtLSG1-2. This gene was highly expressed in regions of auxin accumulation. Ribosome profiling revealed that a loss of function of AtLSG1-2 led to decreased levels of monosomes, further demonstrating its role in ribosome biogenesis. Quantitative proteomics showed that the expression of certain proteins involved in ribosome biogenesis was differentially regulated, indicating that ribosome biogenesis processes were impaired in the mutant. Further investigations showed that an AtLSG1-2 deficiency caused the alteration of auxin distribution, response, and transport in plants. It is concluded that AtLSG1-2 is integral to ribosome biogenesis, consequently affecting auxin homeostasis and plant development.

The Arabidopsis gene DIG6 encodes a large 60S subunit nuclear export GTPase 1 that is involved in ribosome biogenesis and affects multiple auxin-regulated development processes.

Posttranslational lipid modifications mediate the membrane attachment of Rab GTPases, facilitating their function in regulating intracellular vesicular trafficking. In Arabidopsis, most Rab GTPases have two C-terminal cysteines and potentially can be double-geranylgeranylated by heterodimeric Rab geranylgeranyltransferases (Rab-GGTs). Genes encoding two putative α subunits and two putative β subunits of Rab-GGTs have been annotated in the Arabidopsis thaliana genome, but little is known about Rab-GGT activity in Arabidopsis. In this study, we demonstrate that four different heterodimers can be formed between putative Arabidopsis Rab-GGT α subunits RGTA1/RGTA2 and β subunits RGTB1/RGTB2, but only RGTA1·RGTB1 and RGTA1·RGTB2 exhibit bona fide Rab-GGT activity, and they are biochemically redundant in vitro. We hypothesize that RGTA2 function might be disrupted by a 12-amino acid insertion in a conserved motif. We present evidence that Arabidopsis Rab-GGTs may have preference for prenylation of C-terminal cysteines in particular positions. We also demonstrate that Arabidopsis Rab-GGTs can not only prenylate a great variety of Rab GTPases in the presence of Rab escort protein but, unlike Rab-GGT in yeast and mammals, can also prenylate certain non-Rab GTPases independently of Rab escort protein. Our findings may help to explain some of the phenotypes of Arabidopsis protein prenyltransferase mutants.

Arabidopsis Rab Geranylgeranyltransferases Demonstrate Redundancy and Broad Substrate Specificity in Vitro.

Early-diverging land plants such as mosses are known for their outstanding abilities to grow in various terrestrial habitats, incorporating tremendous structural and physiological innovations, as well as many lineage-specific genes. How these genes and functional innovations evolved remains unclear. In this study, we show that a dual-coding gene YAN/AltYAN in the moss Physcomitrella patens evolved from a pre-existing hemerythrin gene. Experimental evidence indicates that YAN/AltYAN is involved in fatty acid and lipid metabolism, as well as oil body and wax formation. Strikingly, both the recently evolved dual-coding YAN/AltYAN and the pre-existing hemerythrin gene might have similar physiological effects on oil body biogenesis and dehydration resistance. These findings bear important implications in understanding the mechanisms of gene origination and the strategies of plants to fine-tune their adaptation to various habitats.

Gene refashioning through innovative shifting of reading frames in mosses.

We have developed a multienzyme functionalized membrane reactor for bioconversion of lignin model compound involving enzymatic catalysis. Layer-by-layer approach was used to immobilize three different enzymes (glucose oxidase, peroxidase and laccase) into pH-responsive membranes. This novel membrane reactor couples the in situ generation of hydrogen peroxide (by glucose oxidase) to oxidative conversion of a lignin model compound, guaiacylglycerol-B-guaiacylether (GGE). Preliminary investigation of the efficacy of these functional membranes towards GGE degradation is demonstrated under convective flow mode. Over 90% of the initial feed could be degraded with the multienzyme immobilized membranes at a residence time of approximately 22 seconds. GGE conversion product analysis revealed formation of oligomeric oxidation products with peroxidase, which might be potential hazard to membrane bioreactors. These oxidation products could be further degraded by laccase enzymes in the multienzymatic membranes explaining the potential of multienzyme membrane reactors. The multienzyme incorporated membrane reactors were active for about a month time of storage at 4 °C, and retention of activity was demonstrated after repetitive use.

Multienzyme immobilized polymeric membrane reactor for transformation of lignin model compound.

Plant colonization of land has been intimately associated with mycorrhizae or mycorrhizae-like fungi. Despite the pivotal role of fungi in plant adaptation, it remains unclear whether and how gene acquisition following fungal interaction might have affected the development of land plants. Here we report a macro2 domain gene in bryophytes that is likely derived from Mucoromycota, a group that includes some mycorrhizae-like fungi found in the earliest land plants. Experimental and transcriptomic evidence suggests that this macro2 domain gene in the moss Physcomitrella patens, PpMACRO2, is important in epigenetic modification, stem cell function, cell reprogramming and other processes. Gene knockout and over-expression of PpMACRO2 significantly change the number and size of gametophores. These findings provide insights into the role of fungal association and the ancestral gene repertoire in the early evolution of land plants.

A mycorrhizae-like gene regulates stem cell and gametophore development in mosses.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Author Correction: A mycorrhizae-like gene regulates stem cell and gametophore development in mosses.

Bio-inspired Topographically Mediated Surfaces (TMSs) based on high aspect ratio nanostructures have recently been attracting significant attention due to their pronounced antimicrobial properties by mechanically disrupting cellular processes. However, scalability of such surfaces is often greatly limited, as most of them rely on micro/nanoscale fabrication techniques. In this report, a cost-effective, scalable, and versatile approach of utilizing diamond nanotechnology for producing TMSs, and using them for limiting the spread of emerging infectious diseases, is introduced. Specifically, diamond-based nanostructured coatings are synthesized in a single-step fabrication process with a densely packed, needle- or spike-like morphology. The antimicrobial proprieties of the diamond nanospike surface are qualitatively and quantitatively analyzed and compared to other surfaces including copper, silicon, and even other diamond surfaces without the nanostructuring. This surface is found to have superior biocidal activity, which is confirmed via scanning electron microscopy images showing definite and widespread destruction of E. coli cells on the diamond nanospike surface. Consistent antimicrobial behavior is also observed on a sample prepared seven years prior to testing date.

A scalable approach to topographically mediated antimicrobial surfaces based on diamond.

A complete picture of how signaling pathways lead to multicellularity is largely unknown. Previously, we generated mutations in a protein prenylation enzyme, GGB, and showed that it is essential for maintaining multicellularity in the moss Physcomitrium patens. Here, we show that ROP GTPases act as downstream factors that are prenylated by GGB and themselves play an important role in the multicellularity of P. patens. We also show that the loss of multicellularity caused by the suppression of GGB or ROP GTPases is due to uncoordinated cell expansion, defects in cell wall integrity and the disturbance of the directional control of cell plate orientation. Expressing prenylatable ROP in the ggb mutant not only rescues multicellularity in protonemata but also results in development of gametophores. Although the prenylation of ROP is important for multicellularity, a higher threshold of active ROP is required for gametophore development. Thus, our results suggest that ROP activation via prenylation by GGB is a key process at both cell and tissue levels, facilitating the developmental transition from one dimension to two dimensions and to three dimensions in P. patens.

The cellular function of ROP GTPase prenylation is important for multicellularity in the moss Physcomitrium patens.

This paper describes the fabrication of cicada-wing-inspired antimicrobial surfaces using Glancing Angle Deposition (GLAD). From the study of an annual cicada (Neotibicen Canicularis, also known as dog-day cicada) in North America, it is found that the cicada wing surfaces are composed of unique three-dimensional (3D) nanofeature arrays, which grant them extraordinary properties including antimicrobial (antifouling) and antireflective. However, the morphology of these 3D nanostructures imposes challenges in artificially synthesizing the structures by utilizing and scaling up the template area from nature. From the perspective of circumventing the difficulties of creating 3D nanofeature arrays with top-down nanofabrication techniques, this paper introduces a nanofabrication process that combines bottom-up steps: self-assembled nanospheres are used as the bases of the features, while sub-100 nm pillars are grown on top of the bases by GLAD. Scanning electron micrographs show the resemblance of the synthesized cicada wing mimicry samples to the actual cicada wings, both quantitatively and qualitatively. The synthetic mimicry samples are hydrophobic with a water contact angle of 125˚. Finally, the antimicrobial properties of the mimicries are validated by showing flat growth curves of Escherichia coli (E. coli) and by direct observation under scanning electron microscopy (SEM). The process is potentially suitable for large-area antimicrobial applications in food and biomedical industries.

Article	Year
Aquaporin 4 molecular mimicry and implications for neuromyelitis optica. Journal of neuroimmunology\| PubMed ID: 23664693	2013
The role of lipid post-translational modification in plant developmental processes. Frontiers in plant science\| PubMed ID: 24600462	2014
Turning moss into algae: prenylation targets in Physcomitrella patens. Plant signaling & behavior\| PubMed ID: 25763501	2014
The Arabidopsis gene DIG6 encodes a large 60S subunit nuclear export GTPase 1 that is involved in ribosome biogenesis and affects multiple auxin-regulated development processes. Journal of experimental botany\| PubMed ID: 26272902	2015
Arabidopsis Rab Geranylgeranyltransferases Demonstrate Redundancy and Broad Substrate Specificity in Vitro. The Journal of biological chemistry\| PubMed ID: 26589801	2016
Gene refashioning through innovative shifting of reading frames in mosses. Nature communications\| PubMed ID: 29674719	2018
Multienzyme immobilized polymeric membrane reactor for transformation of lignin model compound. Polymers\| PubMed ID: 30719335	2018
A mycorrhizae-like gene regulates stem cell and gametophore development in mosses. Nature communications\| PubMed ID: 32332755	2020
Author Correction: A mycorrhizae-like gene regulates stem cell and gametophore development in mosses. Nature communications\| PubMed ID: 32499564	2020
A scalable approach to topographically mediated antimicrobial surfaces based on diamond. Journal of nanobiotechnology\| PubMed ID: 34963490	2021
The cellular function of ROP GTPase prenylation is important for multicellularity in the moss Physcomitrium patens. Development (Cambridge, England)\| PubMed ID: 35660859	2022
Bio-inspired antimicrobial surfaces fabricated by glancing angle deposition. Scientific reports\| PubMed ID: 36604529	2023

Mark P. Running

Biography

JoVE Journal Publications

Other Publications