Quantum enhanced superresolution microscopy (Conference Presentation)

ABSTRACT Autophagy is a catabolic process widely conserved among eukaryotes that permits the rapid degradation of unwanted proteins and organelles through the lysosomal pathway. This mechanism involves the formation of a double-membrane structure called the autophagosome that sequesters cellular components to be degraded. To orchestrate this process, yeasts and animals rely on a conserved set of autophagy-related proteins (ATGs). Key among these factors is ATG8, a cytoplasmic protein that is recruited to nascent autophagosomal membranes upon the induction of autophagy. Toxoplasma gondii is a potentially harmful human pathogen in which only a subset of ATGs appears to be present. Although this eukaryotic parasite seems able to generate autophagosomes upon stresses such as nutrient starvation, the full functionality and biological relevance of a canonical autophagy pathway are as yet unclear. Intriguingly, in T. gondii, ATG8 localizes to the apicoplast under normal intracellular growth conditions. The apicoplast is a nonphotosynthetic plastid enclosed by four membranes resulting from a secondary endosymbiosis. Using superresolution microscopy and biochemical techniques, we show that TgATG8 localizes to the outermost membrane of this organelle. We investigated the unusual function of TgATG8 at the apicoplast by generating a conditional knockdown mutant. Depletion of TgATG8 led to rapid loss of the organelle and subsequent intracellular replication defects, indicating that the protein is essential for maintaining apicoplast homeostasis and thus for survival of the tachyzoite stage. More precisely, loss of TgATG8 led to abnormal segregation of the apicoplast into the progeny because of a loss of physical interactions of the organelle with the centrosomes. IMPORTANCE By definition, autophagy is a catabolic process that leads to the digestion and recycling of eukaryotic cellular components. The molecular machinery of autophagy was identified mainly in model organisms such as yeasts but remains poorly characterized in phylogenetically distant apicomplexan parasites. We have uncovered an unusual function for autophagy-related protein ATG8 in Toxoplasma gondii: TgATG8 is crucial for normal replication of the parasite inside its host cell. Seemingly unrelated to the catabolic autophagy process, TgATG8 associates with the outer membrane of the nonphotosynthetic plastid harbored by the parasite called the apicoplast, and there it plays an important role in the centrosome-driven inheritance of the organelle during cell division. This not only reveals an unexpected function for an autophagy-related protein but also sheds new light on the division process of an organelle that is vital to a group of important human and animal pathogens.

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Dual-Color 3D Superresolution Microscopy by Combined Spectral-Demixing and Biplane Imaging

Biophysical Journal ◽

10.1016/j.bpj.2015.05.026 ◽

2015 ◽

Vol 109 (1) ◽

pp. 3-6 ◽

Cited By ~ 15

Author(s):

Christian M. Winterflood ◽

Evgenia Platonova ◽

David Albrecht ◽

Helge Ewers

Keyword(s):

Superresolution Microscopy ◽

Dual Color

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Synaptic abnormalities and cytoplasmic glutamate receptor aggregates in contactin associated protein-like 2/Caspr2 knockout neurons

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1423205112 ◽

2015 ◽

Vol 112 (19) ◽

pp. 6176-6181 ◽

Cited By ~ 61

Author(s):

Olga Varea ◽

Maria Dolores Martin-de-Saavedra ◽

Katherine J. Kopeikina ◽

Britta Schürmann ◽

Hunter J. Fleming ◽

...

Keyword(s):

Ampa Receptors ◽

Knockout Mice ◽

Copy Number Variations ◽

Spine Density ◽

Structured Illumination ◽

Single Nucleotide Variants ◽

Glutamatergic Synapses ◽

Superresolution Microscopy ◽

Synaptic Markers ◽

Cellular Phenotypes

Central glutamatergic synapses and the molecular pathways that control them are emerging as common substrates in the pathogenesis of mental disorders. Genetic variation in the contactin associated protein-like 2 (CNTNAP2) gene, including copy number variations, exon deletions, truncations, single nucleotide variants, and polymorphisms have been associated with intellectual disability, epilepsy, schizophrenia, language disorders, and autism. CNTNAP2, encoded by Cntnap2, is required for dendritic spine development and its absence causes disease-related phenotypes in mice. However, the mechanisms whereby CNTNAP2 regulates glutamatergic synapses are not known, and cellular phenotypes have not been investigated in Cntnap2 knockout neurons. Here we show that CNTNAP2 is present in dendritic spines, as well as axons and soma. Structured illumination superresolution microscopy reveals closer proximity to excitatory, rather than inhibitory synaptic markers. CNTNAP2 does not promote the formation of synapses and cultured neurons from Cntnap2 knockout mice do not show early defects in axon and dendrite outgrowth, suggesting that CNTNAP2 is not required at this stage. However, mature neurons from knockout mice show reduced spine density and levels of GluA1 subunits of AMPA receptors in spines. Unexpectedly, knockout neurons show large cytoplasmic aggregates of GluA1. Here we characterize, for the first time to our knowledge, synaptic phenotypes in Cntnap2 knockout neurons and reveal a novel role for CNTNAP2 in GluA1 trafficking. Taken together, our findings provide insight into the biological roles of CNTNAP2 and into the pathogenesis of CNTNAP2-associated neuropsychiatric disorders.

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Correlative live-cell and superresolution microscopy reveals cargo transport dynamics at microtubule intersections

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1219206110 ◽

2013 ◽

Vol 110 (9) ◽

pp. 3375-3380 ◽

Cited By ~ 124

Author(s):

Štefan Bálint ◽

Ione Verdeny Vilanova ◽

Ángel Sandoval Álvarez ◽

Melike Lakadamyali

Keyword(s):

Live Cell ◽

Superresolution Microscopy ◽

Transport Dynamics ◽

Cargo Transport

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Dynamic nanoscale morphology of the ER surveyed by STED microscopy

The Journal of Cell Biology ◽

10.1083/jcb.201809107 ◽

2018 ◽

Vol 218 (1) ◽

pp. 83-96 ◽

Cited By ~ 39

Author(s):

Lena K. Schroeder ◽

Andrew E.S. Barentine ◽

Holly Merta ◽

Sarah Schweighofer ◽

Yongdeng Zhang ◽

...

Keyword(s):

Living Cells ◽

Stimulated Emission ◽

Membrane Structures ◽

Spatiotemporal Resolution ◽

Sted Microscopy ◽

Superresolution Microscopy ◽

Structure And Dynamics ◽

First Time ◽

Nanoscale Dynamics ◽

Conventional Light Microscopy

The endoplasmic reticulum (ER) is composed of interconnected membrane sheets and tubules. Superresolution microscopy recently revealed densely packed, rapidly moving ER tubules mistaken for sheets by conventional light microscopy, highlighting the importance of revisiting classical views of ER structure with high spatiotemporal resolution in living cells. In this study, we use live-cell stimulated emission depletion (STED) microscopy to survey the architecture of the ER at 50-nm resolution. We determine the nanoscale dimensions of ER tubules and sheets for the first time in living cells. We demonstrate that ER sheets contain highly dynamic, subdiffraction-sized holes, which we call nanoholes, that coexist with uniform sheet regions. Reticulon family members localize to curved edges of holes within sheets and are required for their formation. The luminal tether Climp63 and microtubule cytoskeleton modulate their nanoscale dynamics and organization. Thus, by providing the first quantitative analysis of ER membrane structure and dynamics at the nanoscale, our work reveals that the ER in living cells is not limited to uniform sheets and tubules; instead, we suggest the ER contains a continuum of membrane structures that includes dynamic nanoholes in sheets as well as clustered tubules.

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Phase Mask-Based Multimodal Superresolution Microscopy

Photonics ◽

10.3390/photonics4030039 ◽

2017 ◽

Vol 4 (4) ◽

pp. 39 ◽

Cited By ~ 4

Author(s):

Ryan Beams ◽

Jeremiah Woodcock ◽

Jeffrey Gilman ◽

Stephan Stranick

Keyword(s):

Phase Mask ◽

Superresolution Microscopy

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Extraction of accurate cytoskeletal actin velocity distributions from noisy measurements

10.1101/2020.08.13.247304 ◽

2020 ◽

Author(s):

Cayla M. Miller ◽

Elgin Korkmazhan ◽

Alexander R. Dunn

Keyword(s):

Single Molecule ◽

Actin Filaments ◽

Dynamical Behavior ◽

Jump Process ◽

Pairwise Distance ◽

Actin Dynamics ◽

Cell Cortex ◽

Superresolution Microscopy ◽

Distance Distributions ◽

Primary Fibroblasts

Dynamic remodeling of the actin cytoskeleton allows cells to migrate, change shape, and exert mechanical forces on their surroundings. How the complex dynamical behavior of the cytoskeleton arises from the interactions of its molecular components remains incompletely understood. Tracking the movement of individual actin filaments in living cells can in principle provide a powerful means of addressing this question. However, single-molecule fluorescence imaging measurements that could provide this information are limited by low signal-to-noise ratios, with the result that the localization errors for individual fluorophore fiducials attached to filamentous (F)-actin are comparable to the distances traveled by actin filaments between measurements. In this study we tracked the movement F-actin labeled with single-molecule densities of the fluorogenic label SiR-actin in primary fibroblasts and endothelial cells. We then used a Bayesian statistical approach to estimate true, underlying actin filament velocity distributions from the tracks of individual actin-associated fluorophores along with quantified localization uncertainties. This analysis approach is broadly applicable to inferring statistical pairwise distance distributions arising from noisy point localization measurements such as occur in superresolution microscopy. We found that F-actin velocity distributions were better described by a statistical jump process, in which filaments exist in mechanical equilibria punctuated by abrupt, jump-like movements, than by models incorporating combinations of diffusive motion and drift. A model with exponentially distributed time- and length-scales for filament jumps recapitulated F-actin velocity distributions measured for the cell cortex, integrin-based adhesions, and actin stress fibers, indicating that a common physical model can potentially describe F-actin dynamics in a variety of cellular contexts.

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