Localized Plasmonic Structured Illumination Microscopy Using Hybrid Inverse Design.

Super-resolution fluorescence imaging has offered unprecedented insights and revolutionized our understanding of biology. In particular, localized plasmonic structured illumination microscopy (LPSIM) achieves video-rate super-resolution imaging with ∼50 nm spatial resolution by leveraging subdiffraction-limited nearfield patterns generated by plasmonic nanoantenna arrays. However, the conventional trial-and-error design process for LPSIM arrays is time-consuming and computationally intensive, limiting the exploration of optimal designs. Here, we propose a hybrid inverse design framework combining deep learning and genetic algorithms to refine LPSIM arrays. A population of designs is evaluated using a trained convolutional neural network, and a multiobjective optimization method optimizes them through iteration and evolution. Simulations demonstrate that the optimized LPSIM substrate surpasses traditional substrates, exhibiting higher reconstruction accuracy, robustness against noise, and increased tolerance for fewer measurements. This framework not only proves the efficacy of inverse design for tailoring LPSIM substrates but also opens avenues for exploring new plasmonic nanostructures in imaging applications.

Super-resolution microscopy to study membrane nanodomains and transport mechanisms in the plasma membrane.

Biological membranes are complex, heterogeneous, and dynamic systems that play roles in the compartmentalization and protection of cells from the environment. It is still a challenge to elucidate kinetics and real-time transport routes for molecules through biological membranes in live cells. Currently, by developing and employing super-resolution microscopy; increasing evidence indicates channels and transporter nano-organization and dynamics within membranes play an important role in these regulatory mechanisms. Here we review recent advances and discuss the major advantages and disadvantages of using super-resolution microscopy to investigate protein organization and transport within plasma membranes.

Visualizing highly bright and uniform cellular ultrastructure by expansion-microscopy with tetrahedral DNA nanostructures.

Expansion Microscopy (ExM) is a widely used super-resolution technique that enables imaging of structures beyond the diffraction limit of light. However, ExM suffers from weak labeling signals and expansion distortions, limiting its applicability. Here, we present an innovative approach called Tetrahedral DNA nanostructure Expansion Microscopy (TDN-ExM), addressing these limitations by using tetrahedral DNA nanostructures (TDNs) for fluorescence labeling. Our approach demonstrates a 3- to 10-fold signal amplification due to the multivertex nature of TDNs, allowing the modification of multiple dyes. Previous studies have confirmed minimal distortion on a large scale, and our strategy can reduce the distortion at the ultrastructural level in samples because it does not rely on anchoring agents and is not affected by digestion. This results in a brighter fluorescence, better uniformity, and compatibility with different labeling strategies and optical super-resolution technologies. We validated the utility of TDN-ExM by imaging various biological structures with improved resolutions and signal-to-noise ratios.

Enhanced Performance of the Optimized Dye CF583R in Direct Stochastic Optical Reconstruction Microscopy of Active Zones in Drosophila Melanogaster.

Super-resolution single-molecule localization microscopy (SMLM) of presynaptic active zones (AZs) and postsynaptic densities contributed to the observation of protein nanoclusters that are involved in defining functional characteristics and in plasticity of synaptic connections. Among SMLM techniques, direct stochastic optical reconstruction microscopy (dSTORM) depends on organic fluorophores that exert high brightness and reliable photoswitching. While multicolor imaging is highly desirable, the requirements necessary for high-quality dSTORM make it challenging to identify combinations of equally performing, spectrally separated dyes. Red-excited carbocyanine dyes, e.g., Alexa Fluor 647 (AF647) or Cy5, are currently regarded as "gold standard" fluorophores for dSTORM imaging. However, a recent study introduced a set of chemically modified rhodamine dyes, including CF583R, that promise to display similar performance in dSTORM. In this study, we defined CF583R's performance compared to AF647 and CF568 based on a nanoscopic analysis of Bruchpilot (Brp), a nanotopologically well-characterized scaffold protein at Drosophila melanogaster AZs. We demonstrate equal suitability of AF647, CF568 and CF583R for basal AZ morphometry, while in Brp subcluster analysis CF583R outperforms CF568 and is on par with AF647. Thus, the AF647/CF583R combination will be useful in future dSTORM-based analyses of AZs and other subcellularly located marker molecules and their role in physiological and pathophysiological contexts.

DivIVA controls the dynamics of septum splitting and cell elongation in Streptococcus pneumoniae.

Bacterial shape and division rely on the dynamics of cell wall assembly, which involves regulated synthesis and cleavage of the peptidoglycan. In ovococci, these processes are coordinated within an annular mid-cell region with nanometric dimensions. More precisely, the cross-wall synthesized by the divisome is split to generate a lateral wall, whose expansion is insured by the insertion of the so-called peripheral peptidoglycan by the elongasome. Septum cleavage and peripheral peptidoglycan synthesis are, thus, crucial remodeling events for ovococcal cell division and elongation. The structural DivIVA protein has long been known as a major regulator of these processes, but its mode of action remains unknown. Here, we integrate click chemistry-based peptidoglycan labeling, direct stochastic optical reconstruction microscopy, and in silico modeling, as well as epifluorescence and stimulated emission depletion microscopy to investigate the role of DivIVA in Streptococcus pneumoniae cell morphogenesis. Our work reveals two distinct phases of peptidoglycan remodeling during the cell cycle that are differentially controlled by DivIVA. In particular, we show that DivIVA ensures homogeneous septum cleavage and peripheral peptidoglycan synthesis around the division site and their maintenance throughout the cell cycle. Our data additionally suggest that DivIVA impacts the contribution of the elongasome and class A penicillin-binding proteins to cell elongation. We also report the position of DivIVA on either side of the septum, consistent with its known affinity for negatively curved membranes. Finally, we take the opportunity provided by these new observations to propose hypotheses for the mechanism of action of this key morphogenetic protein.IMPORTANCEThis study sheds light on fundamental processes governing bacterial growth and division, using integrated click chemistry, advanced microscopy, and computational modeling approaches. It addresses cell wall synthesis mechanisms in the opportunistic human pathogen Streptococcus pneumoniae, responsible for a range of illnesses (otitis, pneumonia, meningitis, septicemia) and for one million deaths every year worldwide. This bacterium belongs to the morphological group of ovococci, which includes many streptococcal and enterococcal pathogens. In this study, we have dissected the function of DivIVA, which is a structural protein involved in cell division, morphogenesis, and chromosome partitioning in Gram-positive bacteria. This work unveils the role of DivIVA in the orchestration of cell division and elongation along the pneumococcal cell cycle. It not only enhances our understanding of how ovoid bacteria proliferate but also offers the opportunity to consider how DivIVA might serve as a scaffold and sensor for particular membrane regions, thereby participating in various cell cycle processes.

A novel super-resolution microscopy platform for cutaneous alpha-synuclein detection in Parkinson's disease.

Alpha-synuclein (aSyn) aggregates in the central nervous system are the main pathological hallmark of Parkinson's disease (PD). ASyn aggregates have also been detected in many peripheral tissues, including the skin, thus providing a novel and accessible target tissue for the detection of PD pathology. Still, a well-established validated quantitative biomarker for early diagnosis of PD that also allows for tracking of disease progression remains lacking. The main goal of this research was to characterize aSyn aggregates in skin biopsies as a comparative and quantitative measure for PD pathology. Using direct stochastic optical reconstruction microscopy (dSTORM) and computational tools, we imaged total and phosphorylated-aSyn at the single molecule level in sweat glands and nerve bundles of skin biopsies from healthy controls (HCs) and PD patients. We developed a user-friendly analysis platform that offers a comprehensive toolkit for researchers that combines analysis algorithms and applies a series of cluster analysis algorithms (i.e., DBSCAN and FOCAL) onto dSTORM images. Using this platform, we found a significant decrease in the ratio of the numbers of neuronal marker molecules to phosphorylated-aSyn molecules, suggesting the existence of damaged nerve cells in fibers highly enriched with phosphorylated-aSyn molecules. Furthermore, our analysis found a higher number of aSyn aggregates in PD subjects than in HC subjects, with differences in aggregate size, density, and number of molecules per aggregate. On average, aSyn aggregate radii ranged between 40 and 200 nm and presented an average density of 0.001-0.1 molecules/nm2. Our dSTORM analysis thus highlights the potential of our platform for identifying quantitative characteristics of aSyn distribution in skin biopsies not previously described for PD patients while offering valuable insight into PD pathology by elucidating patient aSyn aggregation status.

Dissecting the role of SMN multimerization in its dissociation from the Cajal body using harmine as a tool compound.

SMN, linked to spinal muscular atrophy, is a key component of the Gemin complex essential for snRNP assembly. Following initial snRNP assembly in the cytoplasm, both snRNPs and SMN migrate to the nucleus and associate with Cajal bodies, where final snRNP maturation occurs. It is assumed that SMN must be free from the Cajal bodies for continuous snRNP biogenesis. Previous observation of the SMN granules docked in CB suggests the existence of a separation mechanism. However, the precise processes that regulate the spatial separation of SMN-complexes from Cajal bodies remain unclear. Here we employed a super-resolution microscope alongside the beta-carboline alkaloid harmine, which disrupted the Cajal body in a reversible manner. Upon removal of harmine, SMN and Coilin first appear as small, interconnected condensates. The SMN condensates mature into spheroidal structures encircled by Coilin, eventually segregating into distinct condensates. Expression of a multimerization-deficient SMN mutant leads to enlarged, atypical Cajal bodies where SMN is unable to segregate into separate condensates. These findings underscore the importance of multimerization in facilitating the segregation of SMN from Coilin within Cajal bodies.

The conformation of tetraspanins CD53 and CD81 differentially affects their nanoscale organization and interaction with their partners.

Tetraspanins, including CD53 and CD81, are four-transmembrane proteins that affect the membrane organization to regulate cellular processes including migration, proliferation, and signaling. However, it is unclear how the organizing function of tetraspanins is regulated at the molecular level. Here, we investigated whether recently proposed "open" and "closed" conformations of tetraspanins regulate the nanoscale organization of the plasma membrane of B cells. We generated conformational mutants of CD53 (F44E) and CD81 (4A, E219Q) that represent the "closed" and "open" conformation, respectively. Surface expression of these CD53 and CD81 mutants was comparable to that of WT protein. Localization of mutant tetraspanins into nanodomains was visualized by super-resolution direct stochastic optical reconstruction microscopy. Whereas the size of these nanodomains was unaffected by conformation, the clustered fraction of "closed" CD53 was higher and of "open" CD81 lower than respective WT protein. In addition, KO cells lacking CD53 showed an increased likelihood of clustering of its partner CD45. Interestingly, "closed" CD53 interacted more with CD45 than WT CD53. Absence of CD81 lowered the cluster size of its partner CD19 and "closed" CD81 interacted less with CD19 than WT CD81, but "open" CD81 did not affect CD19 interaction. However, none of the tetraspanin conformations made significant impact on the nanoscale organization of their partners CD19 or CD45. Taken together, conformational mutations of CD53 and CD81 differentially affect their nanoscale organization, but not the organization of their partner proteins. This study improves the molecular insight into cell surface nanoscale organization by tetraspanins.

SV2A controls the surface nanoclustering and endocytic recruitment of Syt1 during synaptic vesicle recycling.

Following exocytosis, the recapture of plasma membrane-stranded vesicular proteins into recycling synaptic vesicles (SVs) is essential for sustaining neurotransmission. Surface clustering of vesicular proteins has been proposed to act as a 'pre-assembly' mechanism for endocytosis that ensures high-fidelity retrieval of SV cargo. Here, we used single-molecule imaging to examine the nanoclustering of synaptotagmin-1 (Syt1) and synaptic vesicle protein 2A (SV2A) in hippocampal neurons. Syt1 forms surface nanoclusters through the interaction of its C2B domain with SV2A, which are sensitive to mutations in this domain (Syt1K326A/K328A) and SV2A knockdown. SV2A co-clustering with Syt1 is reduced by blocking SV2A's cognate interaction with Syt1 (SV2AT84A). Surprisingly, impairing SV2A-Syt1 nanoclustering enhanced the plasma membrane recruitment of key endocytic protein dynamin-1, causing accelerated Syt1 endocytosis, altered intracellular sorting and decreased trafficking of Syt1 to Rab5-positive endocytic compartments. Therefore, SV2A and Syt1 are segregated from the endocytic machinery in surface nanoclusters, limiting dynamin recruitment and negatively regulating Syt1 entry into recycling SVs.

In Vivo Monitoring of Nucleophagy in Caenorhabditis elegans.

The autophagy-lysosomal pathway enables the controlled degradation of cellular contents. Nucleophagy is the selective autophagic recycling of nuclear components upon delivery to the lysosome. Although methods to monitor and quantify autophagy as well as selective types of autophagy have been developed and implemented in cells and in vivo, methods monitoring nucleophagy remain scarce. Here, we describe a procedure to monitor the autophagic engagement of an endogenous nuclear envelope component, i.e., ANC-1, the nematode homologue of the mammalian Nesprins in vivo, utilizing super-resolution microscopy.

Quantitative Super-Resolution Imaging of ER-Phagy Initiation in Cells.

Selective autophagy of the endoplasmic reticulum (ER-phagy) is a mechanism that is necessary for degrading damaged ER components and preventing cells from experiencing ER stress. Various ER-phagy receptors orchestrate this process by building protein assemblies with dedicated functions. In order to understand the molecular building principles of ER-phagy, it is important to reveal the assembly of ER-phagy receptors in a temporal and functional context. However, direct visualization is hampered by the diffraction limit in light microscopy. Super-resolution microscopy (SRM) can bypass this limitation and resolve single proteins and nanoscale protein clusters in cells. In particular, DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) is a powerful technology that can resolve individual protein clusters in cells and provide information on their molecular composition. Here, we report a step-by-step protocol on how to utilize DNA-PAINT to perform super-resolution imaging of ER-phagy receptors in fixed cells. In addition, we provide a detailed explanation of image generation, cluster analysis, and molecular quantification.

Posterior approach to correct for focal plane offsets in lattice light-sheet structured illumination microscopy.

Significance: Lattice light-sheet structured illumination microscopy (latticeSIM) has proven highly effective in producing three-dimensional images with super resolution rapidly and with minimal photobleaching. However, due to the use of two separate objectives, sample-induced aberrations can result in an offset between the planes of excitation and detection, causing artifacts in the reconstructed images. Aim: We introduce a posterior approach to detect and correct the axial offset between the excitation and detection focal planes in latticeSIM and provide a method to minimize artifacts in the reconstructed images. Approach: We utilized the residual phase information within the overlap regions of the laterally shifted structured illumination microscopy information components in frequency space to retrieve the axial offset between the excitation and the detection focal planes in latticeSIM. Results: We validated our technique through simulations and experiments, encompassing a range of samples from fluorescent beads to subcellular structures of adherent cells. We also show that using transfer functions with the same axial offset as the one present during data acquisition results in reconstructed images with minimal artifacts and salvages otherwise unusable data. Conclusion: We envision that our method will be a valuable addition to restore image quality in latticeSIM datasets even for those acquired under non-ideal experimental conditions.

DNA-PAINT Probe Modifications Support High-Resolution Imaging with Shorter Binding Domains.

DNA-based Points Accumulation for Imaging in Nanoscale Topography (DNA-PAINT) is an effective super resolution microscopy technique, and its optimization is key to improve nanoscale detection. The state-of-the-art improvements that are at the base of this optimization have been first routinely validated on DNA nanostructure devices before being tested on biological samples. This allows researchers to finely tune DNA-PAINT imaging features in a more controllable in vitro environment. Dye-labeled oligonucleotide probes with short hybridization domains can expand DNA-PAINT's detection by targeting short nucleotide sequences and improving resolution, speed, and multiplexing. However, developing these probes is challenging as their brief bound state makes them difficult to capture under routine imaging conditions. To extend dwell binding times and promote duplex stability, we introduced structural and chemical modifications to our imager probes. The modifications included mini-hairpins and/or Bridged Nucleic Acids (BNA); both of which increase the thermomechanical stability of a DNA duplex. Using this approach we demonstrate DNA-PAINT imaging with approximately 5 nm resolution using a 4-nucleotide hybridization domain that is 43% shorter than previously reported probes. Imager probes with such short hybridization domains are key for improving detection on DNA nanostructure devices because they have the capability to target a larger number of binding domains per localization unit. This is essential for metrology applications such as Nucleic Acid Memory (NAM) where the information density is dependent on the binding site length. The selected imager probes reported here present imaging resolution equivalent to current state-of-the-art DNA-PAINT probes, creating a strategy to image shorter DNA domains for nanoscience and nanotechnology alike.

Metamaterial-Assisted Illumination Nanoscopy with Exceptional Axial Resolution.

Recent advancements in optical metamaterials have opened new possibilities in the exciting field of super-resolution microscopies. The far-field metamaterial-assisted illumination nanoscopies (MAINs) have, very recently, enhanced the lateral resolution to one-fifteenth of the optical wavelength. However, the axial localization accuracy of fluorophores in the MAINs remains rarely explored. Here, a MAIN with a nanometer-scale axial localization accuracy is demonstrated by monitoring the distance-dependent photobleaching dynamics of the fluorophores on top of an organic hyperbolic metamaterial (OHM) substrate under a wide-field single-objective microscope. With such a regular experimental configuration, 3D imaging of various biological samples with the resolution of ≈40 nm in the lateral dimensions and ≈5 nm in the axial dimension is realized. The demonstrated imaging modality enables the resolution of the 3D morphology of nanoscopic cellular structures with a significantly simplified experimental setup.

Advancing cellular insights: Super-resolution STORM imaging of cytoskeletal structures in human stem and cancer cells.

Fluorescence microscopy is an important tool for cell biology and cancer research. Present-day approach of implementing advanced optical microscopy methods combined with immunofluorescence labelling of specific proteins in cells is now able to deliver optical super-resolution up to ∼25 nm. Here we perform super-resolved imaging using standard immunostaining protocol combined with easy stochastic optical reconstruction microscopy (easySTORM) to observe structural differences of two cytoskeleton elements, actin and tubulin in three different cell types namely human bone marrow-derived mesenchymal stem cells (MSCs), human glioblastoma (U87MG) and breast cancer (MDAMB-231) cells. The average width of the actin bundle obtained from STORM images of stem cells is observed to be larger than the same for U87MG and MDAMB-231 cells. No significant difference is however noticed in the width of the tubulin within the same cells. We also study the functional effect on the 2D migration potential of MDAMB-231 cells silenced for NICD1 and ß-catenin. Although similar migration speed is observed for cells with the above two conditions compared to their control cells, easySTORM images show that widths of the actin in MDAMB-231 cells in ß-catenin silenced is significantly lower than the same in control cells. Such minute differences however are not observable in widefield images. The outcome of our easySTORM investigation should benefit the researchers carrying out detailed investigations of the cellular structure and potential therapeutic applications.

Calibrating Fluorescence Microscopy With 3D-Speckler (3D Fluorescence Speckle Analyzer).

Fluorescence microscopy has been widely accessible and indispensable in cell biology research. This technique enables researchers to label targets, ranging from individual entities to multiple groups, with fluorescent markers. It offers precise determinations of localization, size, and shape, along with accurate quantifications of fluorescence signal intensities. Furthermore, an ideal fluorescence microscope can achieve approximately 250 nm in lateral and 600 nm in axial resolution. Despite its integral role in these measurements, the calibration of fluorescence microscopes is often overlooked. This protocol introduces the use of 3D-Speckler (3D fluorescence speckle analyzer), a semi-automated software tool we have recently developed, for calibrating fluorescence microscopy. Calibration of fluorescence microscopy includes determining resolution limits, validating accuracy in size measurements, evaluating illumination flatness, and determining chromatic aberrations. 3D-Speckler is user-friendly and enables precise quantification of fluorescence puncta, including nanoscale 2D/3D particle size, precise locations, and intensity information. By utilizing multispectral fluorescence beads of known sizes alongside 3D-Speckler, the software can effectively calibrate imaging systems. We emphasize the importance of routine calibration for imaging systems to maintain their integrity and reproducibility, ensuring accurate quantification. This protocol provides a detailed step-by-step guide on using 3D-Speckler to calibrate imaging systems. Key features • Semi-automated particle detection. • Accurate three-dimensional measurement of fluorescent particle sizes. • High-precision three-dimensional localization of fluorescent particles. • Precision analysis of point spread function and chromatic aberration in fluorescence microscopy.

Using Localization Microscopy to Quantify Calcium Channels at Presynaptic Boutons.

Calcium channels at synaptic boutons are critical for synaptic function, but their number and distribution are poorly understood. This gap in knowledge is primarily due to the resolution limits of fluorescence microscopy. In the last decade, the diffraction limit of light was surpassed, and fluorescent molecules can now be localized with nanometer precision. Concurrently, new gene editing strategies allowed direct tagging of the endogenous calcium channel genes-expressed in the correct cells and at physiological levels. Further, the repurposing of self-labeling enzymes to attach fluorescent dyes to proteins improved photon yields enabling efficient localization of single molecules. Here, we describe tagging strategies, localization microscopy, and data analysis for calcium channel localization. In this case, we are imaging calcium channels fused with SNAP or HALO tags in live anesthetized C. elegans nematodes, but the analysis is relevant for any super-resolution preparations. We describe how to process images into localizations and protein clusters into confined nanodomains. Finally, we discuss strategies for estimating the number of calcium channels present at synaptic boutons. Key features • Super-resolution imaging of live anesthetized C. elegans. • Three-color super-resolution reconstruction of synapses. • Nanodomains and the distribution of proteins. • Quantification of the number of proteins at synapses from single-molecule localization data.

Super-resolution imaging reveals nucleolar encapsulation by single-stranded DNA.

In eukaryotic cell nuclei, specific sets of proteins gather in nuclear bodies and facilitate distinct genomic processes. The nucleolus, a nuclear body, functions as a factory for ribosome biogenesis by accumulating constitutive proteins, such as RNA polymerase I and nucleophosmin 1 (NPM1). Although in vitro assays have suggested the importance of liquid-liquid phase separation (LLPS) of constitutive proteins in nucleolar formation, how the nucleolus is structurally maintained with intranuclear architecture remains unknown. This study revealed that the nucleolus is encapsulated by single-stranded (ss) DNA-based molecular complex inside the cell nucleus. Super-resolution lattice-structured illumination microscopy (lattice-SIM) showed high abundance of ssDNA beyond the "outer shell" of the nucleolus. Nucleolar disruption and the release of NPM1 were caused by in situ digestion of ssDNA, suggesting that ssDNA has a structural role in nucleolar encapsulation. Furthermore, we identified that ssDNA forms molecular complex with histone H1 for nucleolar encapsulation. Thus, this study illustrates how ssDNA-based molecular complex uphold the structural integrity of nuclear bodies to coordinate genomic processes such as gene transcription and replication.

Identification of z-axis filopodia in growth cones using super-resolution microscopy.

A growth cone is a highly motile tip of an extending axon that is crucial for neural network formation. Three-dimensional-structured illumination microscopy, a type of super-resolution light microscopy with a resolution that overcomes the optical diffraction limitation (ca. 200 nm) of conventional light microscopy, is well suited for studying the molecular dynamics of intracellular events. Using this technique, we discovered a novel type of filopodia distributed along the z-axis ("z-filopodia") within the growth cone. Z-filopodia were typically oriented in the direction of axon growth, not attached to the substratum, protruded spontaneously without microtubule invasion, and had a lifetime that was considerably shorter than that of conventional filopodia. Z-filopodia formation and dynamics were regulated by actin-regulatory proteins, such as vasodilator-stimulated phosphoprotein, fascin, and cofilin. Chromophore-assisted laser inactivation of cofilin induced the rapid turnover of z-filopodia. An axon guidance receptor, neuropilin-1, was concentrated in z-filopodia and was transported together with them, whereas its ligand, semaphorin-3A, was selectively bound to them. Membrane domains associated with z-filopodia were also specialized and resembled those of lipid rafts, and their behaviors were closely related to those of neuropilin-1. The results suggest that z-filopodia have unique turnover properties, and unlike xy-filopodia, do not function as force-generating structures for axon extension.

Mild Hyperthermia-Induced Thermogenesis in the Endoplasmic Reticulum Defines Stress Response Mechanisms.

Previous studies reported that a mild, non-protein-denaturing, fever-like temperature increase induced the unfolded protein response (UPR) in mammalian cells. Our dSTORM super-resolution microscopy experiments revealed that the master regulator of the UPR, the IRE1 (inositol-requiring enzyme 1) protein, is clustered as a result of UPR activation in a human osteosarcoma cell line (U2OS) upon mild heat stress. Using ER thermo yellow, a temperature-sensitive fluorescent probe targeted to the endoplasmic reticulum (ER), we detected significant intracellular thermogenesis in mouse embryonic fibroblast (MEF) cells. Temperatures reached at least 8 °C higher than the external environment (40 °C), resulting in exceptionally high ER temperatures similar to those previously described for mitochondria. Mild heat-induced thermogenesis in the ER of MEF cells was likely due to the uncoupling of the Ca2+/ATPase (SERCA) pump. The high ER temperatures initiated a pronounced cytosolic heat-shock response in MEF cells, which was significantly lower in U2OS cells in which both the ER thermogenesis and SERCA pump uncoupling were absent. Our results suggest that depending on intrinsic cellular properties, mild hyperthermia-induced intracellular thermogenesis defines the cellular response mechanism and determines the outcome of hyperthermic stress.