In vivo analysis of endocytic and biosynthetic transport to the plant vacuole

The plasma membrane forms the interaction site between a cell and its environment. The proteins in the plasma membrane, namely translocators and receptors, allow for the exchange of nutrients and information. This, however, urges for a stringent regulation of these proteins. One mechanism to control their amount is to transport them into the lytic vacuole via the endocytic pathway. The degradative function of vacuoles depends on proteolytic enzymes, which reach that very organelle through a different route. They are synthesized in the endoplasmic reticulum and transported via the endomembrane system. Vacuolar transport of those soluble proteins depends on sorting receptors, separate them from secretory cargo. To gain a deeper understanding on the trafficking of membrane-bound and soluble cargo to the vacuole, we aimed at characterizing the machinery mediating those processes. For this, we employed nanobody-epitope interactions to create intra-cellular setups, which enabled us to perform transport- and interaction-analyses of proteins via confocal microscopy. We revealed that “Vacuolar Sorting Receptors” (VSRs) interact with their ligands in the endoplasmic reticulum and the Golgi apparatus, but not in the trans-Golgi network and the multivesicular body, by performing “Fluorescent Lifetime Imaging to measure Förster Resonance Energy Transfer” (FRET-FLIM; Künzl et al., 2016). In order to create the reporters for the compartment-specific FRET-FLIM measurements, we linked the ligand binding domain of the VSRs to marker-proteins via a nanobody-epitope interaction. We demonstrated that VSRs do indeed recycle and identified the cis-Golgi as the destination of their retrograde transport (Früholz et al., 2018). These discoveries were based on the combination of two nanobody-epitope pairs. We used those for post-translational labelling and trapping of vacuolar sorting receptors. Concerning the machinery mediating the transport of to-be-degraded plasma membrane proteins to the vacuole, we analyzed the “Endosomal Sorting Complex Required For Transport II” (ESCRT-II). Here, we employed FRETFLIM to show that “Vacuolar Protein Sorting 22” (VPS22), 25 and 36 interact to form this specific complex. We pushed the limits of nanobody-based approaches by employing membrane-anchored nanobodies in order to import the method of co-immune precipitation into living cells. This enabled us to perform in vivo studies, which showed that ESCRT-II contains two VPS25 moieties (Fäßler et al., prepared manuscript)

Cryo-electron tomography structure of Arp2/3 complex in cells reveals new insights into the branch junction

The actin-related protein (Arp)2/3 complex nucleates branched actin filament networks pivotal for cell migration, endocytosis and pathogen infection. Its activation is tightly regulated and involves complex structural rearrangements and actin filament binding, which are yet to be understood. Here, we report a 9.0 Å resolution structure of the actin filament Arp2/3 complex branch junction in cells using cryo-electron tomography and subtomogram averaging. This allows us to generate an accurate model of the active Arp2/3 complex in the branch junction and its interaction with actin filaments. Notably, our model reveals a previously undescribed set of interactions of the Arp2/3 complex with the mother filament, significantly different to the previous branch junction model. Our structure also indicates a central role for the ArpC3 subunit in stabilizing the active conformation

Deciphering the molecular mechanisms of actin cytoskeleton regulation in cell migration using cryo-EM

The actin cytoskeleton plays a key role in cell migration and cellular morphodynamics in most eukaryotes. The ability of the actin cytoskeleton to assemble and disassemble in a spatiotemporally controlled manner allows it to form higher-order structures, which can generate forces required for a cell to explore and navigate through its environment. It is regulated not only via a complex synergistic and competitive interplay between actin-binding proteins (ABP), but also by filament biochemistry and filament geometry. The lack of structural insights into how geometry and ABPs regulate the actin cytoskeleton limits our understanding of the molecular mechanisms that define actin cytoskeleton remodeling and, in turn, impact emerging cell migration characteristics. With the advent of cryo-electron microscopy (cryo-EM) and advanced computational methods, it is now possible to define these molecular mechanisms involving actin and its interactors at both atomic and ultra-structural levels in vitro and in cellulo. In this review, we will provide an overview of the available cryo-EM methods, applicable to further our understanding of the actin cytoskeleton, specifically in the context of cell migration. We will discuss how these methods have been employed to elucidate ABP- and geometry-defined regulatory mechanisms in initiating, maintaining, and disassembling cellular actin networks in migratory protrusions

3D printed cell culture grid holders for improved cellular specimen preparation in cryo-electron microscopy

Cryo-electron microscopy (cryo-EM) of cellular specimens provides insights into biological processes and structures within a native context. However, a major challenge still lies in the efficient and reproducible preparation of adherent cells for subsequent cryo-EM analysis. This is due to the sensitivity of many cellular specimens to the varying seeding and culturing conditions required for EM experiments, the often limited amount of cellular material and also the fragility of EM grids and their substrate. Here, we present low-cost and reusable 3D printed grid holders, designed to improve specimen preparation when culturing challenging cellular samples directly on grids. The described grid holders increase cell culture reproducibility and throughput, and reduce the resources required for cell culturing. We show that grid holders can be integrated into various cryo-EM workflows, including micro-patterning approaches to control cell seeding on grids, and for generating samples for cryo-focused ion beam milling and cryo-electron tomography experiments. Their adaptable design allows for the generation of specialized grid holders customized to a large variety of applications

A Rapid and Simple Method for Microscopy-Based Stomata Analyses

There are two major methodical approaches with which changes of status in stomatal pores are addressed: indirectly by measurement of leaf transpiration, and directly by measurement of stomatal apertures. Application of the former method requires special equipment, whereas microscopic images are utilized for the direct measurements. Due to obscure visualization of cell boundaries in intact leaves, a certain degree of invasive leaf manipulation is often required. Our aim was to develop a protocol based on the minimization of leaf manipulation and the reduction of analysis completion time, while still producing consistent results. We applied rhodamine 6G staining of Arabidopsis thaliana leaves for stomata visualization, which greatly simplifies the measurement of stomatal apertures. By using this staining protocol, we successfully conducted analyses of stomatal responses in Arabidopsis leaves to both closure and opening stimuli. We performed long-term monitoring of living stomata and were able to document the same leaf before and after treatment. Moreover, we developed a protocol for rapid-fixation of epidermal peels, which enables high throughput data analysis. The described method allows analysis of stomatal apertures with minimal leaf manipulation and usage of the same leaf for sequential measurements, and will facilitate the analysis of several lines in parallel

Computational toolbox for ultrastructural quantitative analysis of filament networks in cryo-ET data

A precise quantitative description of the ultrastructural characteristics underlying biological mechanisms is often key to their understanding. This is particularly true for dynamic extra- and intracellular filamentous assemblies, playing a role in cell motility, cell integrity, cytokinesis, tissue formation and maintenance. For example, genetic manipulation or modulation of actin regulatory proteins frequently manifests in changes of the morphology, dynamics, and ultrastructural architecture of actin filament-rich cell peripheral structures, such as lamellipodia or filopodia. However, the observed ultrastructural effects often remain subtle and require sufficiently large datasets for appropriate quantitative analysis. The acquisition of such large datasets has been enabled by recent advances in high-throughput cryo-electron tomography (cryo-ET) methods. This also necessitates the development of complementary approaches to maximize the extraction of relevant biological information. We have developed a computational toolbox for the semi-automatic quantification of segmented and vectorized fila- mentous networks from pre-processed cryo-electron tomograms, facilitating the analysis and cross-comparison of multiple experimental conditions. GUI-based components simplify the processing of data and allow users to obtain a large number of ultrastructural parameters describing filamentous assemblies. We demonstrate the feasibility of this workflow by analyzing cryo-ET data of untreated and chemically perturbed branched actin filament networks and that of parallel actin filament arrays. In principle, the computational toolbox presented here is applicable for data analysis comprising any type of filaments in regular (i.e. parallel) or random arrangement. We show that it can ease the identification of key differences between experimental groups and facilitate the in-depth analysis of ultrastructural data in a time-efficient manner

Cryo-electron tomography of the onion cell wall shows bimodally oriented cellulose fibers and reticulated homogalacturonan networks

One hallmark of plant cells is their cell wall. They protect cells against the environment and high turgor and mediate morphogenesis through the dynamics of their mechanical and chemical properties. The walls are a complex polysaccharidic structure. Although their biochemical composition is well known, how the different components organize in the volume of the cell wall and interact with each other is not well understood and yet is key to the wall’s mechanical properties. To investigate the ultrastructure of the plant cell wall, we imaged the walls of onion (Allium cepa) bulbs in a near-native state via cryo-focused ion beam milling (cryo-FIB milling) and cryo-electron tomography (cryo-ET). This allowed the high-resolution visualization of cellulose fibers in situ. We reveal the coexistence of dense fiber fields bathed in a reticulated matrix we termed “meshing,” which is more abundant at the inner surface of the cell wall. The fibers adopted a regular bimodal angular distribution at all depths in the cell wall and bundled according to their orientation, creating layers within the cell wall. Concomitantly, employing homogalacturonan (HG)-specific enzymatic digestion, we observed changes in the meshing, suggesting that it is—at least in part—composed of HG pectins. We propose the following model for the construction of the abaxial epidermal primary cell wall: the cell deposits successive layers of cellulose fibers at −45° and +45° relative to the cell’s long axis and secretes the surrounding HG-rich meshing proximal to the plasma membrane, which then migrates to more distal regions of the cell wall

Changes in stomatal apertures in <i>Arabidopsis</i> leaves.

<p>Stomata closure in response to 10 μM ABA and 100 μM H₂O₂ in Col-0, <i>ahk5-1</i> and AHK5 overexpressor lines (a) and Ws-4 and <i>ahk5-3</i> mutant line (b). Three fully expanded leaves at a similar developmental stage (one leaf per plant) per treatment were analyzed for each line. A single asterisk indicates a significant difference to corresponding mock-treated leaves (*, 0.01 < p ≤ 0.05), two asterisks depict a very significant difference (**, 0.001 < p ≤ 0.01), and three asterisks indicate an extremely significant difference to corresponding mock-treated leaves (***, p ≤ 0.001). (c,d) Comparison of stomatal movements in rhodamine 6G pre-stained (before treatment with hormones) and post-stained <i>Arabidopsis</i> leaves. (c) Stomatal apertures in leaves treated with 10 μM ABA or 5 μM IAA for 2 h. <i>pre-st</i>.: pre-stained leaves, <i>post-st</i>.: post-stained leaves. (d) Stomatal apertures in leaves treated with 10 μM ABA for 30 min and 1 h. A total of six leaves (c) and nine leaves (d) at a similar developmental stage (one leaf per plant) were used for the analyses. A very significant difference between leaves before treatment (0 h) and after treatment is indicated by two asterisks (**, 0.001 < p ≤ 0.01), an extremely significant difference is indicated by three asterisks (***, p ≤ 0.001).</p