AbstractWe are interested in the mechanisms by which cells generate and maintain their complex architectures. A major structural element in cells are lipid membranes, which define both the external cell shape as well as forming subcellular compartments, such as organelles. Fixation methods that maintain membranes in their native states are thus critical for examining cells using transmission electron microscopy (TEM). One such method is high pressure freezing (HPF), in which rapid freezing under high pressures is used to prevent the formation of water ice crystals that otherwise disrupt cellular architecture. To examine the ultrastructure of terminal cells, a component of the insect respiratory system, we have developed an HPF technique for preservation of larval Drosophila. In addition, we have adapted our HPF method to fix larvae in such a way that fluorescence of transgenic markers is maintained. This so called correlative light /electron microscopy (CLEM) procedure allows us to identify cells using light microscopy and then perform TEM on the same samples.

We are now using our technique to characterize Drosophila mutants that have defects in specific cells within tracheal terminal cells. Terminal cells show a complex branched morphology and contain a subcellular, membrane-bound lumen. In our mutants, lumen formation fails. Ordinarily, such cells are very hard to identify by TEM, since the presence of the lumen is the defining characteristic of a terminal cell. Here we describe using a CLEM procedure to identify mutant cells in sections, based on their expression of a cell-type specific fluorescent marker, and then to characterize cell ultrastructure using TEM.

(A) Nucleus of an epidermal cell. Nuclear envelope membranes are well preserved and nuclear pores can be identified (arrowheads). ncl = nucleolus.

(B) Epidermal cell (epi) with finger like projections extending from the apical surface (higher magnification shown in inset). pro = chitinous pro-cuticle.

(C) Microvilli (vil) on the gut apical surface and mitochondria (mit) within the cell cytoplasm are clearly visible. lum = lumen of gut.

(D) Higher magnification view of the gut cytoplasm showing rough endoplasmic reticulum (RER) and mitochondria (mit).

(E) Cross section of nerve bundle with tightly fasciculated neuronal processes.

(F) Glial cell ensheathing a nerve bundle.

Scale bars = 200nm.

(A) A single larval tracheal terminal cell expressing GFP and visualized by light microscopy. Each terminal cell develops a branched network of cytoplasmic processes extending from the cell body (indicated with an arrow). Each branch is a tiny tube and contains a submicron, membrane bound and air-filled lumen. Scale bar = 50µm.

The goal of CLEM procedures is to fix samples for TEM analysis in such a way that expression of fluorescence marker proteins is conserved. This allows cells of interest to be identified by light microscopy and the ultrastructure of these cells to them be assessed by EM. We have adapted our HPF technique such that activity of the fluorescent reporter protein DsRed is conserved. Critical to this work was testing combinations of membrane fixatives and embedding media in which both fluorescence and preservation of cellular ultrastructure is maintained. We found fixation with 0.001% osmium tetroxide plus 0.1% of potassium permanganate and embedding in the hydrophobic resin glycidyl methacrylate (GM) leads to a optimal conservation of both fluorescence and ultrastructure. (A-D) show examples of ultrastructural preservation. (A) Epidermal cell nucleus. ncl = nucleolus; pro = overlying chitinous procuticle; (B) ensheathed nerve bundle; (C) unicellular tracheal cell showing taenidial folds (arrow); (D) muscle cell and mitochondria (magnified in inset). (E) Intact Drosophila larvae expressing DsRed throughout the tracheal system fixed for EM sectioning and embedded in a GM block. (F) DsRed expression in ultrathin section observed using TIRF microscopy and (F’) adjacent section observed by TEM. Four terminal branch profiles (labeled 1-4) can be observed

in the sections. (G) High magnification of terminal branch profile #4. Apical lumen membrane (arrowheads) and taenidial folds (arrows) have been preserved. Also observable is rough ER (asterisks). This section is at a site of branching, thus two lumens are present. Scale bars: A-D, 1µm; E, 0.5mm; F, 5µm; G, 500nm.

(A & B) Cross sections of developing tracheal terminal branches lacking mature air-filled lumens. A large, membrane bound intracellular compartment containing vesicles (arrowheads in insets) appears to be a lumen formation intermediate. In (A) the compartment contains a subcompartment that appears clear of cytoplasmic components (asterisk). In (B) a rudimentary chitin matrix is forming inside the compartment (arrow). (C) Serial section EM on the branch lumen shown in (A) reveals the compartment is completely bounded by membrane and roughly spheroidal. (D) Diagram of subcellular lumen intermediate . Grey: tracheal branch; green: compartment limiting membrane; red: vesicles and lumen precursor within the limiting membrane. Scale bars = 200nm.

(B & C) TEM analysis of terminal cell branches in HPF-fixed larvae: (B) cross section; (C) longitudinal section. The lumen of the air-filled tube (lum) is lined by a elaborate, ridged chitinous matrix, called taenidial folds (asterisks). Terminal branches frequently run between the epidermis (epi) and muscle (mus) cells. µ = longitudinal microtubules. B’ and C’ show schematic representations of the images in B & C. Scale bars = 200nm.

Examples of cells in a Drosophila larva fixed using HPF and embedded in epoxy resin

•We have developed high pressure freezing methods to effectively preserve Drosophila larvae for ultrastructural analysis by TEM

•Using our technique we have analyze development of subcellular lumenogenesis in terminal cells and have identified a novel candidate intermediates in the lumen formation process

•We have developed methods to perform correlative light/electron microscopy on Drosophila larvae and are using these to analyze mutants in which terminal cell lumen formation is abnormal

Analysis of Drosophila tracheal (respiratory) terminal cell ultrastructure by HPF

TEM analysis can be used to determine ultrastructural phenotypes of mutants in which terminal cell lumen formation is abnormal. Terminal cells mutant for either Rbcn-3A (M.M.M. unpublished) or Zpr1 (Ruiz et al., PLoS One Sep. 2012) fail to form air-filled lumens. By light microscopy the phenotypes of these two mutants are indistinguishable. However, by TEM analysis the mutants show distinct cellular defects. Guided by our CLEM approach, we identified mutant terminal cells and examined them for defects in lumen ultrastructure. Lumens in Rbcn-3A mutants contain multiple membranous whorls (magnified on right), suggesting a defect in membrane trafficking processes required to form the apical membrane defining the lumen. The apical luminal membrane in Zpr1 mutants appear to be normal (arrows), but the internal chitinous lining appears to be collapsed, indicating a defect in taenidia formation or luminal gas filling. Scale bars = 200nm.

Acknowledgments We are very grateful to Shigeki Watanabe and Erik Jorgensen (University of Utah) for sharing protocols, reagents, and access to the Zeiss PAL-M microscope. We thank David Belnap and Ed King (University of Utah) for providing us with equipment and the Core Facilities at the University of Utah for funding to attend the Microscopy and Microanalysis meeting.

Ultrastructural characterization of lumen formation mutantsIdentification of a candidate lumen-formation intermediates by TEM analysis

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Linda S. Nikolova and Mark M. MetzsteinUniversity of Utah, Salt Lake City, Utah

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Correlative light/electron microscopy (CLEM)

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Development of high-pressure freezing (HPF) and correlative light/electron microscopy (CLEM) for Drosophila larvae

Highlights