Bachelor of Science, Goteborgs Universitet (2009)
Master of Science, Goteborgs Universitet (2010)
Doctor of Philosophy, Goteborgs Universitet (2015)
William Moerner, Postdoctoral Faculty Sponsor
Novel fibrillar structure in the inversin compartment of primary cilia revealed by 3D single-molecule super-resolution microscopy.
Molecular biology of the cell
Primary cilia in many cell types contain a peri-axonemal sub-compartment called the inversin compartment. Four proteins have been found to assemble within the inversin compartment: INVS, ANKS6, NEK8, and NPHP3. The function of the inversin compartment is unknown, but it appears to be critical for normal development including left-right asymmetry and renal tissue homeostasis. Here we combine super-resolution imaging of human RPE1 cells, a classic model for studying primary cilia in vitro, with a genetic dissection of the protein-protein binding relationships that organize compartment assembly to develop a new structural model. We observe that INVS is the core structural determinant of a compartment composed of novel fibril-like substructures, which we identify here by 3D single-molecule super-resolution imaging. We find that NEK8 and ANKS6 depend on INVS for localization to these fibrillar assemblies and that ANKS6-NEK8 density within the compartment is regulated by NEK8. Together, NEK8 and ANKS6 are required downstream of INVS to localize and concentrate NPHP3 within the compartment. In the absence of these upstream components, NPHP3 is redistributed within cilia. These results provide a more detailed structure for the inversin compartment and introduce a new example of a membraneless compartment organized by protein-protein interactions. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text].
View details for DOI 10.1091/mbc.E19-09-0499
View details for PubMedID 31895004
Quantitative Super-Resolution Microscopy of the Mammalian Glycocalyx.
The mammalian glycocalyx is a heavily glycosylated extramembrane compartment found on nearly every cell. Despite its relevance in both health and disease, studies of the glycocalyx remain hampered by a paucity of methods to spatially classify its components. We combine metabolic labeling, bioorthogonal chemistry, and super-resolution localization microscopy to image two constituents of cell-surface glycans, N-acetylgalactosamine (GalNAc) and sialic acid, with 10-20 nm precision in 2D and 3D. This approach enables two measurements: glycocalyx height and the distribution of individual sugars distal from the membrane. These measurements show that the glycocalyx exhibits nanoscale organization on both cell lines and primary human tumor cells. Additionally, we observe enhanced glycocalyx height in response to epithelial-to-mesenchymal transition and to oncogenic KRAS activation. In the latter case, we trace increased height to an effector gene, GALNT7. These data highlight the power of advanced imaging methods to provide molecular and functional insights into glycocalyx biology.
View details for DOI 10.1016/j.devcel.2019.04.035
View details for PubMedID 31105009
Phosphofructokinase controls the acetaldehyde induced phase shift in isolated yeast glycolytic oscillators.
The Biochemical journal
The response of oscillatory systems to external perturbations is crucial for emergent properties such as synchronization and phase locking, and can be quantified in a phase response curve. In individual, oscillating yeast cells, we characterized experimentally the phase response of glycolytic oscillations for external acetaldehyde pulses, and followed the transduction of the perturbation through the system. Subsequently, we analyzed the control of the relevant system components in a detailed mechanistic model. The observed responses are interpreted in terms of the functional coupling and regulation in the reaction network. We find that our model quantitatively predicts the phase dependent phase shift observed in the experimental data. The phase shift is in agreement with an adaptation leading to synchronization with an external signal. Our model analysis establishes that phosphofructokinase plays a key role in the phase shift dynamics as shown in the phase response curve, and adaptation time to external perturbations. Specific mechanism-based interventions, made possible through such analyses of detailed models, can improve upon standard trial and error methods, e.g. melatonin supplementation to overcome jet-lag, which are error prone, specifically, since the effects are phase and dose dependent.
View details for PubMedID 30482792
Studying Glycolytic Oscillations in Individual Yeast Cells by Combining Fluorescence Microscopy with Microfluidics and Optical Tweezers.
Current protocols in cell biology
In this unit, we provide a clear exposition of the methodology employed to study dynamic responses in individual cells, using microfluidics for controlling and adjusting the cell environment, optical tweezers for precise cell positioning, and fluorescence microscopy for detecting intracellular responses. This unit focuses on the induction and study of glycolytic oscillations in single yeast cells, but the methodology can easily be adjusted to examine other biological questions and cell types. We present a step-by-step guide for fabrication of the microfluidic device, for alignment of the optical tweezers, for cell preparation, and for time-lapse imaging of glycolytic oscillations in single cells, including a discussion of common pitfalls. A user who follows the protocols should be able to detect clear metabolite time traces over the course of up to an hour that are indicative of dynamics on the second scale in individual cells during fast and reversible environmental adjustments. © 2018 by John Wiley & Sons, Inc.
View details for PubMedID 30329225
Quantitative super-resolution microscopy reveals the architecture of the mammalian glycocalyx and its changes during cancer progression
AMER CHEMICAL SOC. 2018
View details for Web of Science ID 000447600001117
Light sheet approaches for improved precision in 3D localization-based super-resolution imaging in mammalian cells [Invited]
2018; 26 (10): 13122–47
The development of imaging techniques beyond the diffraction limit has paved the way for detailed studies of nanostructures and molecular mechanisms in biological systems. Imaging thicker samples, such as mammalian cells and tissue, in all three dimensions, is challenging due to increased background and volumes to image. Light sheet illumination is a method that allows for selective irradiation of the image plane, and its inherent optical sectioning capability allows for imaging of biological samples with reduced background, photobleaching, and photodamage. In this review, we discuss the advantage of combining single-molecule imaging with light sheet illumination. We begin by describing the principles of single-molecule localization microscopy and of light sheet illumination. Finally, we present examples of designs that successfully have married single-molecule super-resolution imaging with light sheet illumination for improved precision in mammalian cells.
View details for DOI 10.1364/OE.26.013122
View details for Web of Science ID 000432457600088
View details for PubMedID 29801343
View details for PubMedCentralID PMC6005674
In Situ Imaging of Spatial Organization of Accessible Chromatin at the Nanoscale with ATAC-see and Single-Molecule Super-Resolution Fluorescence Microscopy
CELL PRESS. 2018: 539A
View details for Web of Science ID 000430563200448
3D Single-Molecule Super-Resolution Microscopy in Mammalian Cells Using a Tilted Light Sheet
CELL PRESS. 2018: 14A
View details for Web of Science ID 000429315800076
Tilted Light Sheet Microscopy with 3D Point Spread Functions for Single-Molecule Super-Resolution Imaging in Mammalian Cells.
Proceedings of SPIE--the International Society for Optical Engineering
To obtain a complete picture of subcellular nanostructures, cells must be imaged with high resolution in all three dimensions (3D). Here, we present tilted light sheet microscopy with 3D point spread functions (TILT3D), an imaging platform that combines a novel, tilted light sheet illumination strategy with engineered long axial range point spread functions (PSFs) for low-background, 3D super localization of single molecules as well as 3D super-resolution imaging in thick cells. TILT3D is built upon a standard inverted microscope and has minimal custom parts. The axial positions of the single molecules are encoded in the shape of the PSF rather than in the position or thickness of the light sheet, and the light sheet can therefore be formed using simple optics. The result is flexible and user-friendly 3D super-resolution imaging with tens of nm localization precision throughout thick mammalian cells. We validated TILT3D for 3D super-resolution imaging in mammalian cells by imaging mitochondria and the full nuclear lamina using the double-helix PSF for single-molecule detection and the recently developed Tetrapod PSF for fiducial bead tracking and live axial drift correction. We envision TILT3D to become an important tool not only for 3D super-resolution imaging, but also for live whole-cell single-particle and single-molecule tracking.
View details for PubMedID 29681676
3D single-molecule super-resolution microscopy with a tilted light sheet
2018; 9: 123
Tilted light sheet microscopy with 3D point spread functions (TILT3D) combines a novel, tilted light sheet illumination strategy with long axial range point spread functions (PSFs) for low-background, 3D super-localization of single molecules as well as 3D super-resolution imaging in thick cells. Because the axial positions of the single emitters are encoded in the shape of each single-molecule image rather than in the position or thickness of the light sheet, the light sheet need not be extremely thin. TILT3D is built upon a standard inverted microscope and has minimal custom parts. The result is simple and flexible 3D super-resolution imaging with tens of nm localization precision throughout thick mammalian cells. We validate TILT3D for 3D super-resolution imaging in mammalian cells by imaging mitochondria and the full nuclear lamina using the double-helix PSF for single-molecule detection and the recently developed tetrapod PSFs for fiducial bead tracking and live axial drift correction.
View details for PubMedID 29317629
Observation of live chromatin dynamics in cells via 3D localization microscopy using Tetrapod point spread functions
BIOMEDICAL OPTICS EXPRESS
2017; 8 (12): 5735–48
We report the observation of chromatin dynamics in living budding yeast (Saccharomyces cerevisiae) cells, in three-dimensions (3D). Using dual color localization microscopy and employing a Tetrapod point spread function, we analyze the spatio-temporal dynamics of two fluorescently labeled DNA loci surrounding the GAL locus. From the measured trajectories, we obtain different dynamical characteristics in terms of inter-loci distance and temporal variance; when the GAL locus is activated, the 3D inter-loci distance and temporal variance increase compared to the inactive state. These changes are visible in spite of the large thermally- and biologically-driven heterogeneity in the relative motion of the two loci. Our observations are consistent with current euchromatin vs. heterochromatin models.
View details for PubMedID 29296501