live-cell-imaging-microtubule-cytoskeleton-micromechanical

Live Cell Imaging of Microtubule Cytoskeleton and Micromechanical Manipulation of the <em>Arabidopsis</em> Shoot Apical Meristem

Understanding cell and tissue level regulation of growth and morphogenesis has been at the forefront of biological research for many decades. Advances in ...

In eukaryotic cells, the actin and microtubule (MT) cytoskeletal networks are dynamic structures that organize intracellular processes and facilitate their rapid reorganization. In plant cells, actin filaments (AFs) and MTs are essential for cell growth and morphogenesis. However, dynamic interactions between these two essential components in live cells have not been explored. Here, we use spinning-disc confocal microscopy to dissect interaction and cooperation between cortical AFs and MTs in Arabidopsis thaliana, utilizing fluorescent reporter constructs for both components. Quantitative analyses revealed altered AF dynamics associated with the positions and orientations of cortical MTs. Reorganization and reassembly of the AF array was dependent on the MTs following drug-induced depolymerization, whereby short AFs initially appeared colocalized with MTs, and displayed motility along MTs. We also observed that light-induced reorganization of MTs occurred in concert with changes in AF behavior. Our results indicate dynamic interaction between the cortical actin and MT cytoskeletons in interphase plant cells.

Live cell imaging reveals structural associations between the actin and microtubule cytoskeleton in Arabidopsis.

The actin and microtubule (MT) cytoskeletons are vital structures for cell growth and development across all species. While individual molecular mechanisms underpinning actin and MT dynamics have been intensively studied, principles that govern the cytoskeleton organization remain largely unexplored. Here, we captured biologically relevant characteristics of the plant cytoskeleton through a network-driven imaging-based approach allowing us to quantitatively assess dynamic features of the cytoskeleton. By introducing suitable null models, we demonstrate that the plant cytoskeletal networks exhibit properties required for efficient transport, namely, short average path lengths and high robustness. We further show that these advantageous features are maintained during temporal cytoskeletal rearrangements. Interestingly, man-made transportation networks exhibit similar properties, suggesting general laws of network organization supporting diverse transport processes. The proposed network-driven analysis can be readily used to identify organizational principles of cytoskeletons in other organisms. 

Quantitative analyses of the plant cytoskeleton reveal underlying organizational principles.

The process of shape change in cells and tissues inevitably involves the modification of structural elements, therefore it is necessary to integrate mechanics with biochemistry to develop a full understanding of morphogenesis. Here, we discuss recent findings on the role of biomechanics and biochemical processes in plant cell growth and development. In particular, we focus on how the plant cytoskeleton components, which are known to regulate morphogenesis, are influenced by biomechanical stress. We also discuss new insights into the role that pectin plays in biomechanics and morphogenesis. Using the jigsaw-shaped pavement cells of the leaf as a case study, we review new findings on the biomechanics behind the morphogenesis of these intricately-shaped cell types. Finally, we summarize important quantitative techniques that has allowed for the testing and the generation of hypotheses that link biomechanics to morphogenesis.

Getting into shape: the mechanics behind plant morphogenesis.

Plastid ribosomes are very similar in structure and function to the ribosomes of their bacterial ancestors. Since ribosome biogenesis is not thermodynamically favorable under biological conditions it requires the activity of many assembly factors. Here we have characterized a homolog of bacterial RsgA in Arabidopsis thaliana and show that it can complement the bacterial homolog. Functional characterization of a strong mutant in Arabidopsis revealed that the protein is essential for plant viability, while a weak mutant produced dwarf, chlorotic plants that incorporated immature pre-16S ribosomal RNA into translating ribosomes. Physiological analysis of the mutant plants revealed smaller, but more numerous, chloroplasts in the mesophyll cells, reduction of chlorophyll a and b, depletion of proplastids from the rib meristem and decreased photosynthetic electron transport rate and efficiency. Comparative RNA sequencing and proteomic analysis of the weak mutant and wild-type plants revealed that various biotic stress-related, transcriptional regulation and post-transcriptional modification pathways were repressed in the mutant. Intriguingly, while nuclear- and chloroplast-encoded photosynthesis-related proteins were less abundant in the mutant, the corresponding transcripts were increased, suggesting an elaborate compensatory mechanism, potentially via differentially active retrograde signaling pathways. To conclude, this study reveals a chloroplast ribosome assembly factor and outlines the transcriptomic and proteomic responses of the compensatory mechanism activated during decreased chloroplast function.

AtRsgA from Arabidopsis thaliana is important for maturation of the small subunit of the chloroplast ribosome.

Plant cells are surrounded by highly dynamic cell walls that play important roles regulating aspects of plant development. Recent advances in visualization and measurement of cell wall properties have enabled accumulation of new data about wall architecture and biomechanics. This has resulted in greater understanding of the dynamics of cell wall deposition and remodeling. The cell wall is the first line of defense against different adverse abiotic and biotic environmental influences. Different abiotic stress conditions such as salinity, drought, and frost trigger production of Reactive Oxygen Species (ROS) which act as important signaling molecules in stress activated cellular responses. Detection of ROS by still-elusive receptors triggers numerous signaling events that result in production of different protective compounds or even cell death, but most notably in stress-induced cell wall remodeling. This is mediated by different plant hormones, of which the most studied are jasmonic acid and brassinosteroids. In this review we highlight key factors involved in sensing, signal transduction, and response(s) to abiotic stress and how these mechanisms are related to cell wall-associated stress acclimatization. ROS, plant hormones, cell wall remodeling enzymes and different wall mechanosensors act coordinately during abiotic stress, resulting in abiotic stress wall acclimatization, enabling plants to survive adverse environmental conditions.

Hitting the Wall-Sensing and Signaling Pathways Involved in Plant Cell Wall Remodeling in Response to Abiotic Stress.

How organisms attain their specific shapes and modify their growth patterns in response to environmental and chemical signals has been the subject of many investigations. Plant cells are at high turgor pressure and are surrounded by a rigid yet flexible cell wall, which is the primary determinant of plant growth and morphogenesis. Cellulose microfibrils, synthesized by plasma membrane-localized cellulose synthase complexes, are major tension-bearing components of the cell wall that mediate directional growth. Despite advances in understanding the genetic and biophysical regulation of morphogenesis, direct studies of cellulose biosynthesis and its impact on morphogenesis of different cell and tissue types are largely lacking. In this study, we took advantage of mutants of three primary cellulose synthase () genes that are involved in primary wall cellulose synthesis. Using field emission scanning electron microscopy, live cell imaging and biophysical measurements, we aimed to understand how the primary wall CESA complex acts during shoot apical meristem development. Our results indicate that cellulose biosynthesis impacts the mechanics and growth of the shoot apical meristem.

Primary wall cellulose synthase regulates shoot apical meristem mechanics and growth.

The cytoskeleton is key to many essential processes in a plant cell, e.g., growth, division, and defense. Contrary to what "skeleton" implies, the cytoskeleton is highly dynamic, and is able to re-organize itself continuously. The advent of live-cell microscopy and the development of genetically encoded fluorophores enabled detailed observation of the organization and dynamics of the cytoskeleton. Despite the biological importance of the cytoskeletal dynamics, quantitative analyses remain laborious endeavors that only a handful of research teams regularly conduct. With this protocol, we provide a standardized step-by-step guide to analyze the dynamics of microtubules. We provide example data and code for post-processing in Fiji that enables researchers to modify and adapt the routine to their needs. More such tools are needed to quantitatively assess the cytoskeleton and thus to better understand cell biology. © 2019 by John Wiley & Sons, Inc.

Quantification of Cytoskeletal Dynamics in Time-Lapse Recordings.

Energy-demanding processes, such as cell growth, migration, and differentiation, are tension modulated, begging the question whether metabolism and mechanical tension are tightly linked. A recent report by Park et al. shows that stiffness in the extracellular matrix (ECM) promotes reorganization of actin, resulting in enhanced glycolysis.

Cytoskeleton Architecture Regulates Glycolysis Coupling Cellular Metabolism to Mechanical Cues.

Tissue folding allows for the development of complex three-dimensional morphologies necessary for various functions. A new study provides novel mechanistic insights linking plant cell wall and hormonal pathways involved in bending of plant tissue through regulation of differential growth.

Article	Year
Live cell imaging reveals structural associations between the actin and microtubule cytoskeleton in Arabidopsis. The Plant cell\| PubMed ID: 21693695	2011
Quantitative analyses of the plant cytoskeleton reveal underlying organizational principles. Journal of the Royal Society, Interface\| PubMed ID: 24920110	2014
Getting into shape: the mechanics behind plant morphogenesis. Current opinion in plant biology\| PubMed ID: 30036706	2018
AtRsgA from Arabidopsis thaliana is important for maturation of the small subunit of the chloroplast ribosome. The Plant journal : for cell and molecular biology\| PubMed ID: 30044525	2018
Hitting the Wall-Sensing and Signaling Pathways Involved in Plant Cell Wall Remodeling in Response to Abiotic Stress. Plants (Basel, Switzerland)\| PubMed ID: 30360552	2018
Primary wall cellulose synthase regulates shoot apical meristem mechanics and growth. Development (Cambridge, England)\| PubMed ID: 31076488	2019
Quantification of Cytoskeletal Dynamics in Time-Lapse Recordings. Current protocols in plant biology\| PubMed ID: 31091014	2019
Cytoskeleton Architecture Regulates Glycolysis Coupling Cellular Metabolism to Mechanical Cues. Trends in biochemical sciences\| PubMed ID: 32345468	2020
Plant Biology: Bending of Plant Organs. Current biology : CB\| PubMed ID: 32369753	2020

Arun Sampathkumar

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