Evolutionarily unique mechanistic framework of clathrin-mediated endocytosis in plants

In plants, clathrin mediated endocytosis (CME) represents the major route for cargo internalisation from the cell surface. It has been assumed to operate in an evolutionary conserved manner as in yeast and animals. Here we report characterisation of ultrastructure, dynamics and mechanisms of plant CME as allowed by our advancement in electron microscopy and quantitative live imaging techniques. Arabidopsis CME appears to follow the constant curvature model and the bona fide CME population generates vesicles of a predominantly hexagonal-basket type; larger and with faster kinetics than in other models. Contrary to the existing paradigm, actin is dispensable for CME events at the plasma membrane but plays a unique role in collecting endocytic vesicles, sorting of internalised cargos and directional endosome movement that itself actively promote CME events. Internalized vesicles display a strongly delayed and sequential uncoating. These unique features highlight the independent evolution of the plant CME mechanism during the autonomous rise of multicellularity in eukaryotes

Evolutionarily unique mechanistic framework of clathrin-mediated endocytosis in plants

In plants, clathrin mediated endocytosis (CME) represents the major route for cargo internalisation from the cell surface. It has been assumed to operate in an evolutionary conserved manner as in yeast and animals. Here we report characterisation of ultrastructure, dynamics and mechanisms of plant CME as allowed by our advancement in electron microscopy and quantitative live imaging techniques. Arabidopsis CME appears to follow the constant curvature model and the bona fide CME population generates vesicles of a predominantly hexagonal-basket type; larger and with faster kinetics than in other models. Contrary to the existing paradigm, actin is dispensable for CME events at the plasma membrane but plays a unique role in collecting endocytic vesicles, sorting of internalised cargos and directional endosome movement that itself actively promote CME events. Internalized vesicles display a strongly delayed and sequential uncoating. These unique features highlight the independent evolution of the plant CME mechanism during the autonomous rise of multicellularity in eukaryotes

Clathrin-mediated Endocytosis with Cell Confinement and during Neutrophil Polarization

Clathrin-mediated endocytosis (CME) is one of the major pathway through which cells internalize nutrients and membrane proteins. It occurs on the membrane via clathrin-coated pits (CCPs). In this thesis, we studied CCPs' behavior when cells are under spatial constraints. In the first two projects, the constraint was applied using micro-contact printing. CCPs presented differential phenotypes on different-sized but the same shaped cells. In particular, CCPs were smaller with larger spreading area. We further showed that this might be due to the higher cortical tension associated with large spreading area. Seeding cells on anisotropic fibronectin patterns, we were able to manipulate where and how long CCPs appear on the cell. Together, these results showed that CCPs' distribution and behavior are regulated by mechanical cues in a cell. In the last project, HL-60 differentiated neutrophils were used as the experimental system. They undergo rapid polarization in the presence of N-formylmethionyl-leucyl-phenylalanine (fMLP), during which cells not only present anisotropic morphology but also have asymetric distribution of cellular structures and signaling molecules. We found that CCPs did not have as polarized distribution upon the stimulation of fMLP, but they revealed differential interation with formyl peptitde recepter, actin, and β-arrestin with and without fMLP. Disruption of CME blocked effective neutrophil polarization as well as major signaling pathways. The results suggest a regulatory role of CME in neutrophil polarization.PHDMechanical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/144015/1/xinyutan_1.pd

Probing Cellular Uptake of Nanoparticles, One at a Time

Advanced fluorescence microscopy is the method of choice to study cellular uptake of nanoparticles with molecular specificity and nanoscale resolution; yet, direct visualization of nanoparticles entry into cells poses severe technical challenges. Here, we have combined super-resolution photoactivation localization microscopy (PALM) with single particle tracking (SPT) to visualize clathrin-mediated endocytosis (CME) of polystyrene nanoparticles at very high spatial and temporal resolution

Super-resolution mapping of receptor engagement during HIV entry

The plasma membrane (PM) serves as a major interface between the cell and extracellular stimuli. Studies indicate that the spatial organisation and dynamics of receptors correlate with the regulation of cellular responses. However, the nanoscale spatial organisation of specific receptor molecules on the surface of cells is not well understood primarily because these spatial events are beyond the resolving power of available tools. With the development in super-resolution microscopy and quantitative analysis approaches, it optimally poises me to address some of these questions. The human immunodeficiency virus type-1 (HIV-1) entry process is an ideal model for studying the functional correlation of the spatial organisation of receptors. The molecular interactions between HIV envelope glycoprotein (Env) and key receptors, CD4 and co-receptor CCR5/CXCR4, on the PM of target cells have been well characterised. However, the spatial organisation that receptors undergo upon HIV-1 binding remains unclear. In this project, I established a Single Molecule Localisation Microscopy (SMLM) based visualisation and quantitative analysis pipeline to characterise CD4 membrane organisation in CD4+ T cells, the main host cell target for HIV-1 infection. I found that prior to HIV engagement, CD4 and CCR5 molecules are organised in small distinct clusters across the PM. Upon HIV-1 engagement, I observed dynamic congregation and subsequent dispersal of virus-associated CD4 clusters within 10min. I further incorporated statistical modelling to show that this reorganisation is not random. This thesis provides one of the first nanoscale imaging and quantitative pipelines for visualising and quantifying membrane receptors. I showed that this quantitative approach provides a robust methodology for understanding the recruitment of HIV-1 receptors before the formation of a fusion pore. This methodology can be applied to the analyses of the nanoscale organisation of PM receptors to link the spatial organisation to function

Optical imaging methods for the study of disease models from the nano to the mesoscale

The visualisation of disease phenotypes allows scientists to study fundamental mechanisms of disease. Optical imaging methods are useful not only to observe anatomical features of biological samples, but also to infer interactions between molecular species using fluorescence labelling. This thesis presents the development of imaging and analysis tools to study biological questions in three models of disease, with samples ranging from the sub-cellular to the organ scale. First, the role of the alpha-synuclein (a-syn) protein, whose dysfunction is a hallmark of Parkinson’s Disease, was studied with respect to vesicle trafficking at the synapse. Synaptic vesicles are ∼40 nm in diameter; imaging vesicles therefore requires methods with resolution below the diffraction limit. Single-molecule localisation microscopy (SMLM), which circumvents the diffraction limit by separating fluorophore emission in time to localise individual molecules in space with ∼20 nm precision, was thus implemented to study a-syn in purified synaptic boutons. A software package was developed to analyse the colocalisation of a-syn with internalised vesicles, and the clustering of a-syn under differing synaptic calcium levels. The colocalisation of a-syn and internalised vesicles was found to be temperature independent, suggesting that a-syn is involved in non-canonical trafficking mechanisms. Ground truth simulations from a synaptosome model were used to benchmark two cluster analysis methods. Both methods applied on the experimental data showed that a-syn becomes less clustered at low synaptic calcium levels. Second, the spatiotemporal association of ESCRT-II, a protein complex whose role in the budding of the human immunodeficiency virus (HIV) was previously considered dispensable, and the HIV polyprotein Gag was studied during viral egress using novel image analysis tools. A nearest-neighbour analysis showed the ESCRT-II protein EAP45 colocalises with Gag similarly to ALIX, a protein well known to be involved in HIV budding. However, upon deletion of EAP45’s N-terminus, its colocalisation with Gag was significantly impaired, highlighting the importance of this EAP45 domain in linking to Gag. Single particle tracking was used to trace the trajectories of EAP45 and Gag in live cells, and an algorithm was developed to visualise the simultaneous motion of two particles; these analyses revealed three types of potential dynamic interaction between EAP45 and Gag. Finally, an open-source instrument to visualise phenotypes from large organs in 3D was developed for the study of chronic obstructive pulmonary disease (COPD) models. The instrument implements Optical Projection Tomography, a technique which can reconstruct cross-sectional slices of a transparent object from its orthographic projections, using off-the- shelf components and novel ImageJ plugins for artefact correction and volume reconstructions. Excised and cleared mouse lungs were imaged in which high order airways can be discerned with 50 μm resolution. The raw lung data, instructions for building the instrument, the free ImageJ plugins, and a detailed software manual are available in an online repository to encourage the widespread use of OPT for imaging large samples.Gates Cambridg

Developing new optical imaging techniques for single particle and molecule tracking in live cells

Differential interference contrast (DIC) microscopy is a far-field as well as wide-field optical imaging technique. Since it is non-invasive and requires no sample staining, DIC microscopy is suitable for tracking the motion of target molecules in live cells without interfering their functions. In addition, high numerical aperture objectives and condensers can be used in DIC microscopy. The depth of focus of DIC is shallow, which gives DIC much better optical sectioning ability than those of phase contrast and dark field microscopies. In this work, DIC was utilized to study dynamic biological processes including endocytosis and intracellular transport in live cells. The suitability of DIC microscopy for single particle tracking in live cells was first demonstrated by using DIC to monitor the entire endocytosis process of one mesoporous silica nanoparticle (MSN) into a live mammalian cell. By taking advantage of the optical sectioning ability of DIC, we recorded the depth profile of the MSN during the endocytosis process. The shape change around the nanoparticle due to the formation of a vesicle was also captured. DIC microscopy was further modified that the sample can be illuminated and imaged at two wavelengths simultaneously. By using the new technique, noble metal nanoparticles with different shapes and sizes were selectively imaged. Among all the examined metal nanoparticles, gold nanoparticles in rod shapes were found to be especially useful. Due to their anisotropic optical properties, gold nanorods showed as diffraction-limited spots with disproportionate bright and dark parts that are strongly dependent on their orientation in the 3D space. Gold nanorods were developed as orientation nanoprobes and were successfully used to report the self-rotation of gliding microtubules on kinesin coated substrates. Gold nanorods were further used to study the rotational motions of cargoes during the endocytosis and intracellular transport processes in live mammalian cells. New rotational information was obtained: (1) during endocytosis, cargoes lost their rotation freedom at the late stage of internalization; (2) cargoes performed train-like motion when they were transported along the microtubule network by motor proteins inside live cells; (3) During the pause stage of fast axonal transport, cargoes were still bound to the microtubule tracks by motor proteins. Total internal reflection fluorescence microscopy (TIRFM) is another non-invasive and far-field optical imaging technique. Because of its near-field illumination mechanism, TIRFM has better axial resolution than epi-fluorescence microscopy and confocal microscopy. In this work, an auto-calibrated, prism type, angle-scanning TIRFM instrument was built. The incident angle can range from subcritical angles to nearly 90y, with an angle interval less than 0.2y. The angle precision of the new instrument was demonstrated through the finding of the surface plasmon resonance (SPR) angle of metal film coated glass slide. The new instrument improved significantly the precision in determining the axial position. As a result, the best obtained axial resolution was ~ 8 nm, which is better than current existing instruments similar in function. The instrument was further modified to function as a pseudo TIRF microscope. The illumination depth can be controlled by changing the incident angle of the excitation laser beam or adjusting the horizontal position of the illumination laser spot on the prism top surface. With the new technique, i.e., variable-illumination-depth pseudo TIRF microscopy, the whole cell body from bottom to top was scanned

ARABIDOPSIS MYOSIN IS INVOLVED IN THE DISTRIBUTION AND DYNAMIC BEHAVIOR OF THE CELLULOSE SYNTHASE COMPLEX

Plant cells are encased in cell walls which are important for the growth and development of the organism. Primary cell wall consists mainly of polysaccharides with cellulose as the most abundant component. In plant cells, cellulose is synthesized by a plasma membrane (PM) localized protein complex called the cellulose synthase complex (CSC). It was previously reported that disrupting normal actin organization resulted in a reduction of cellulose content in Arabidopsis dark-grown seedlings. Furthermore, actin was found to facilitate the delivery of CSC into the PM, and inferred to be involved in endocytosis. As a motor protein that translocates cargo along actin filaments, myosin plays an important role in organelle and vesicle trafficking. However, it is not known whether myosin is involved in regulating cellulose deposition or CSC behavior. Here, we used biochemical analysis to determine the cellulose content in Arabidopsis etiolated seedlings, and found a significantly decreased cellulose content in a myosin xi-1, xi-2, and xi-k triple knockout mutant (xi3KO), indicating that myosin is involved in cellulose deposition. To evaluate the molecular mechanism underlying the role of myosin in CSC trafficking, we characterized and employed a new plant myosin inhibitor

Image Analysis and Platform Development for Automated Phenotyping in Cytomics

This thesis is dedicated to the empirical study of image analysis in HT/HC screen study. Often a HT/HC screening produces extensive amounts that cannot be manually analyzed. Thus, an automated image analysis solution is prior to an objective understanding of the raw image data. Compared to general application domain, the efficiency of HT/HC image analysis is highly subjected to image quantity and quality. Accordingly, this thesis will address two major procedures, namely image segmentation and object tracking, in the image analysis step of HT/HC screen study. Moreover, this thesis focuses on expending generic computer science and machine learning theorems into the design of dedicated algorithms for HT/HC image analysis. Additionally, this thesis exemplifies a practical implementation of image analysis and data analysis workflow via empirical case studies with different image modalities and experiment settings. However, the data analysis theorem will be generally illustrated without further expansions. Finally, the thesis will briefly address supplementary infrastructures for end-user interaction and data visualization.Netherlands Bioinformatics CentreComputer Systems, Imagery and Medi

Characterization of CD93 Diffusion in Human Monocytes and Chinese Hamster Ovary Cells.

The diffusion of CD93, a putative efferocytic receptor, was studied in the membrane of human monocytes and CHO cells using single particle tracking. We found that of CD93 molecules were confined in compartments consistent with actin corrals, while moved freely. Cage effect analysis showed short-lived caging of free CD93 and longer-lasting caging of confined CD93, with smaller corrals resulting in stronger caging. The motion of both free and confined CD93 was found to be consistent with a subdiffusive continuous time random walk, suggesting that CD93 diffusion is affected by several processes. We also sought to develop a total internal reflection fluorescence compatible traction force microscopy substrate intended for use in force characterization in frustrated efferocytosis. TIRF-compatible silicone substrates of a uniform thickness were manufactured successfully, but were found to be unsuitable for reliable force measurements due to their susceptibility to the non-specific adsorption of quantum dots