Ultra-High Resolution 3D Imaging of Whole Cells.

Fluorescence nanoscopy, or super-resolution microscopy, has become an important tool in cell biological research. However, because of its usually inferior resolution in the depth direction (50-80 nm) and rapidly deteriorating resolution in thick samples, its practical biological application has been effectively limited to two dimensions and thin samples. Here, we present the development of whole-cell 4Pi single-molecule switching nanoscopy (W-4PiSMSN), an optical nanoscope that allows imaging of three-dimensional (3D) structures at 10- to 20-nm resolution throughout entire mammalian cells. We demonstrate the wide applicability of W-4PiSMSN across diverse research fields by imaging complex molecular architectures ranging from bacteriophages to nuclear pores, cilia, and synaptonemal complexes in large 3D cellular volumes.

Sphingomyelin is sorted at the trans Golgi network into a distinct class of secretory vesicle.

One of the principal functions of the trans Golgi network (TGN) is the sorting of proteins into distinct vesicular transport carriers that mediate secretion and interorganelle trafficking. Are lipids also sorted into distinct TGN-derived carriers? The Golgi is the principal site of the synthesis of sphingomyelin (SM), an abundant sphingolipid that is transported. To address the specificity of SM transport to the plasma membrane, we engineered a natural SM-binding pore-forming toxin, equinatoxin II (Eqt), into a nontoxic reporter termed Eqt-SM and used it to monitor intracellular trafficking of SM. Using quantitative live cell imaging, we found that Eqt-SM is enriched in a subset of TGN-derived secretory vesicles that are also enriched in a glycophosphatidylinositol-anchored protein. In contrast, an integral membrane secretory protein (CD8α) is not enriched in these carriers. Our results demonstrate the sorting of native SM at the TGN and its transport to the plasma membrane by specific carriers.

Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms.

Newly developed scientific complementary metal-oxide semiconductor (sCMOS) cameras have the potential to dramatically accelerate data acquisition, enlarge the field of view and increase the effective quantum efficiency in single-molecule switching nanoscopy. However, sCMOS-intrinsic pixel-dependent readout noise substantially lowers the localization precision and introduces localization artifacts. We present algorithms that overcome these limitations and that provide unbiased, precise localization of single molecules at the theoretical limit. Using these in combination with a multi-emitter fitting algorithm, we demonstrate single-molecule localization super-resolution imaging at rates of up to 32 reconstructed images per second in fixed and living cells.

Fibroblasts secrete fibronectin under lamellipodia in a microtubule- and myosin II-dependent fashion.

Fibronectin (FN) is an essential structural and regulatory component of the extracellular matrix (ECM), and its binding to integrin receptors supports cell adhesion, migration, and signaling. Here, using live-cell microscopy of fibroblasts expressing FN tagged with a pH-sensitive fluorophore, we show that FN is secreted predominantly at the ventral surface of cells in an integrin-independent manner. Locally secreted FN then undergoes ß1 integrin-dependent fibrillogenesis. We find that the site of FN secretion is regulated by cell polarization, which occurs in bursts under stabilized lamellipodia at the leading edge. Moreover, analysis of FN secretion and focal adhesion dynamics suggest that focal adhesion formation precedes FN deposition and that deposition continues during focal adhesion disassembly. Lastly, we show that the polarized FN deposition in spreading and migrating cells requires both intact microtubules and myosin II-mediated contractility. Thus, while FN secretion does not require integrin binding, the site of exocytosis is regulated by membrane and cytoskeletal dynamics with secretion occurring after new adhesion formation.

A Rab GAP cascade defines the boundary between two Rab GTPases on the secretory pathway.

Membrane traffic along the endocytic and exocytic pathways relies on the appropriate localization and activation of a series of different Rab GTPases. Rabs are activated by specific guanine nucleotide exchange factors (GEFs) and inactivated by GTPase-activating proteins (GAPs). GEF cascades, in which one Rab in its GTP-bound form recruits the GEF that activates the next Rab along the pathway, can account for the sequential activation of a series of Rabs, but it does not explain how the first Rab is inactivated after the next Rab has been activated. We present evidence for a counter-current GAP cascade that serves to restrict the spatial and temporal overlap of 2 Rabs, Ypt1p and Ypt32p, on the exocytic pathway in Saccharomyces cerevisiae. We show that Gyp1p, a GAP for Ypt1p, specifically interacts with Ypt32p, and that this interaction is important for the localization and stability of Gyp1p. Moreover, we demonstrate that, in WT cells, Ypt1p compartments are converted over time into Ypt32p compartments, whereas in gyp1Delta cells there is a significant increase in compartments containing both proteins that reflects a slower transition from Ypt1p to Ypt32p. GEF cascades working in concert with counter-current GAP cascades could generate a programmed series of Rab conversions responsible for regulating the choreography of membrane traffic.

Exocyst complex mediates recycling of internal cilia.

Primary cilia are slender, cellular antennae that sense extracellular stimuli, and their absence or dysfunction plays a role in numerous human diseases. Prior work has indicated a role of the exocyst tethering complex in cilia biogenesis and maintenance,1-6 with the underlying paradigm that the exocyst targets vesicles to the ciliary base to deliver ciliary cargoes.7-9 However, the role of the exocyst vis-à-vis to primary cilia in living cells and during stimulation is unknown. Herein, using advanced imaging and quantitative analysis reveals that serum stimulation increases the exocyst's localization to cilia by three-fold. This serum-stimulated localization is highly dynamic, and FRAP experiments show that exocysts at the cilia are highly mobile (60%-80%). Super resolution imaging reveals that the xocyst extends past the cilia base to the entire ciliary pocket. To visualize cilia exocytosis, we conducted live cell imaging with pH-sensitive cilia reporters in combination with extracellular pH switching. Strikingly, we observed that an exocyst-positive internal cilia fuses with the cell surface. These live cell results support a novel and dynamic role of the exocyst complex in the delivery of internalized cilia to the cell surface. Moreover, they suggest a novel pathway may be used to recycle primary cilia to the cell surface that engages the exocyst in response to stimuli. This new remarkable plasticity in cilia presence on the surface in response to extracellular stimuli suggest new means to potentially modulate cilia signaling.

Global mRNA expression analysis in myosin II deficient strains of Saccharomyces cerevisiae reveals an impairment of cell integrity functions.

BACKGROUND: The Saccharomyces cerevisiae MYO1 gene encodes the myosin II heavy chain (Myo1p), a protein required for normal cytokinesis in budding yeast. Myo1p deficiency in yeast (myo1Delta) causes a cell separation defect characterized by the formation of attached cells, yet it also causes abnormal budding patterns, formation of enlarged and elongated cells, increased osmotic sensitivity, delocalized chitin deposition, increased chitin synthesis, and hypersensitivity to the chitin synthase III inhibitor Nikkomycin Z. To determine how differential expression of genes is related to these diverse cell wall phenotypes, we analyzed the global mRNA expression profile of myo1Delta strains. RESULTS: Global mRNA expression profiles of myo1Delta strains and their corresponding wild type controls were obtained by hybridization to yeast oligonucleotide microarrays. Results for selected genes were confirmed by real time RT-PCR. A total of 547 differentially expressed genes (p < or = 0.01) were identified with 263 up regulated and 284 down regulated genes in the myo1Delta strains. Gene set enrichment analysis revealed the significant over-representation of genes in the protein biosynthesis and stress response categories. The SLT2/MPK1 gene was up regulated in the microarray, and a myo1Deltaslt2Delta double mutant was non-viable. Overexpression of ribosomal protein genes RPL30 and RPS31 suppressed the hypersensitivity to Nikkomycin Z and increased the levels of phosphorylated Slt2p in myo1Delta strains. Increased levels of phosphorylated Slt2p were also observed in wild type strains under these conditions. CONCLUSION: Following this analysis of global mRNA expression in yeast myo1Delta strains, we conclude that 547 genes were differentially regulated in myo1Delta strains and that the stress response and protein biosynthesis gene categories were coordinately regulated in this mutant. The SLT2/MPK1 gene was confirmed to be essential for myo1Delta strain viability, supporting that the up regulated stress response genes are regulated by the PKC1 cell integrity pathway. Suppression of Nikkomycin Z hypersensitivity together with Slt2p phosphorylation was caused by the overexpression of ribosomal protein genes RPL30 and RPS31. These ribosomal protein mRNAs were down regulated in the myo1Delta arrays, suggesting that down regulation of ribosomal biogenesis may affect cell integrity in myo1Delta strains.

2,3-Butanedione monoxime increases sensitivity to Nikkomycin Z in the budding yeast Saccharomyces cerevisiae.

Nikkomycin Z (NZ) is a competitive inhibitor of chitin synthase III in the yeast Saccharomyces cerevisiae. Myosin type II-deficient yeast strains (myo1) display a dramatic reduction in growth when chitin synthase III activity is inhibited by NZ, supporting the contention that actomyosin motility plays an important role in maintaining cell wall integrity. A proposed inhibitor of cortical actin polymerization in vitro, 2,3-butanedione monoxime (BDM), also inhibits growth of wild-type yeast strains at a concentration of 20 mM. In this study, we assayed for potential in vivo interplay between BDM-sensitive cell functions and cell wall chitin synthesis by testing for increased sensitivity to NZ during co-treatment with BDM at sub-inhibitory concentrations. Our results show that BDM can increase the sensitivity of yeast cells to Nikkomycin Z.