End-on imaging: a new perspective on dorsoventral development in Drosophila embryos.

Drosophila ventral furrow formation has frequently been used as a model to study developmentally-regulated cell-shape changes. However, a technique to follow all cellular changes during this process within a single living embryo has been lacking. We describe a novel technique, called "end-on imaging", to collect time-lapse images of transversely mounted living embryos. End-on imaging revealed several new features of dorsoventral development. First, we observed a wave of syncytial nuclear divisions predicting the location of the ventral furrow. Second, we determined that there is a 5-min gap between the end of cellularization and the start of ventral furrow formation, suggesting that the two processes may share the same pool of cytoskeletal components. Lastly, we show that apical-membrane flattening, the first step in ventral furrow formation, is due to the ventral cells pushing against the vitelline membrane, rather than flattening the dome-shaped, apical surfaces of these cells by a pulling or constriction motion.</p

The brain-specific Beta4 subunit downregulates BK channel cell surface expression.

<p>The large-conductance K(+) channel (BK channel) can control neural excitability, and enhanced channel currents facilitate high firing rates in cortical neurons. The brain-specific auxiliary subunit β4 alters channel Ca(++)- and voltage-sensitivity, and β4 knock-out animals exhibit spontaneous seizures. Here we investigate β4's effect on BK channel trafficking to the plasma membrane. Using a novel genetic tag to track the cellular location of the pore-forming BKα subunit in living cells, we find that β4 expression profoundly reduces surface localization of BK channels via a C-terminal ER retention sequence. In hippocampal CA3 neurons from C57BL/6 mice with endogenously high β4 expression, whole-cell BK channel currents display none of the characteristic properties of BKα+β4 channels observed in heterologous cells. Finally, β4 knock-out animals exhibit a 2.5-fold increase in whole-cell BK channel current, indicating that β4 also regulates current magnitude in vivo. Thus, we propose that a major function of the brain-specific β4 subunit in CA3 neurons is control of surface trafficking.</p

Imaging vasculature and lymphatic flow in mice using quantum dots.

Quantum dots are ideal probes for fluorescent imaging of vascular and lymphatic tissues. On injection into appropriate sites, red- and near-infrared-emitting quantum dots provide excellent definition of vasculature, lymphoid organs, and lymph nodes draining both normal tissues and tumors. We detail methods for use with commercially available quantum dots and discuss common difficulties.</p

Genetic ablation of β4 results in larger BK channel currents in CA3 pyramidal neurons.

<p>(A) Fluorescent image of a living slice from a β4−/− knock-out mouse showing GFP-signal in hippocampus. Scale bar: 500 µm. (B) Mean total potassium current from β4 heterozygote (β4+/−) and β4 knock-out (β4−/−) mice. (C) Example paxilline-sensitive BK channel current from representative CA3 neurons in β4+/− and β4−/− mice. (D) Mean amplitude of paxilline-sensitive BK channel current in β4+/− and β4−/− mice.</p

Immunofluorescence reveals that BKα and β4 are substantially localized to the ER and that β4 is difficult to detect on the cell surface.

<p>(A) Immunocytochemistry against fixed, permeabilized HEK-293 cells for the FAP-BKα subunit (anti-BKα: green) and the ER-marker mRFP-KDEL (intrinsic fluorescence; red) in cells transfected with BKα alone. Inset shows a cell with overlapping expression of BKα and mRFP-KDEL with arrows pointing to regions containing only BKα. (B) Quantitation of normalized intensity versus normalized distance from the cell nucleus for anti-BKα (green) and mRFP (red) in transfected cells. (C) As in (A) but for cells expressing BKα (green), β4 (unlabeled; not visualized) and mRFP-KDEL (red). (D) Same as (B) but in cells expressing BKα, β4 and mRFP-KDEL. (E) Localization of the β4 subunit (anti-β4: green) and mRFP- KDEL (intrinsic fluorescence; red) in cells expressing BKα (unlabeled; not visualized), β4 and mRFP-KDEL. Scale bar = 20 µm or 5 µm for inset. (F) As in (B), but for β4 and mRFP-KDEL. (G) Localization of the β4 subunit (anti-β4: green), mRFP-KDEL (intrinsic fluorescence; red) and BKα (anti-α: blue) in transfected, fixed HEK-293 cells expressing BKα, β4, and mRFP-KDEL. (H) Expression pattern of the β4 subunit (anti-β4: green) and mRFP- KDEL (intrinsic fluorescence; red) under non-permeabilized immunohistochemistry conditions. Note that immunohistochemical detection of cell-surface BKα is difficult because of an intracellular epitope for BKα. Scale bar = 10 µm (G,H).</p

Mutation of the C-terminal ER-retention/retrieval sequence in β4 liberates β4 to the surface.

<p>(A) Expression pattern of the cotransfected FAP-BKα (unlabeled; not visualized), β4-Ala mutant (anti-β4 immunocytochemistry: green) and mRFP- KDEL (intrinsic fluorescence; red) in fixed, permeabilized HEK-293 cells. (B) Same as (A) but under non-permeabilized conditions. (C) Same as for (A) but for β4-polySer mutant under permeabilized conditions. (D) Same as (C) but under non-permeabilized conditions. Scale bar = 10 µm (A–D).</p

An ER-retention/retrieval sequence at the C-terminus of β4 inhibits the surface expression of BKα and β4.

<p>(A) Schematic of the mutant β4 constructs showing the C-terminal amino acids. (B) Fluorescence from binding of cell-impermeable dye to cell-surface BKα channels in cells transfected with FAP-BKα (red) and GFP (green). (C) Same as (B) but in cells transfected with FAP-BKα and wild-type β4. (D) Same as (B) but in cells transfected with FAP-BKα and β4-Ala. (E) Same as (B) but in cells transfected with FAP-BKα and β4-polySer. Scale bar = 20 µm (B–E). (F) Mean intensity of surface fluorescence for β4 constructs normalized to fluorescence from cells transfected with FAP-BKα alone. (G) Histogram showing distribution of fluorescence intensity in cells transfected with FAP-BKα alone (red), FAP-BKα+β4-Ala (green) and FAP-BKα+β4-polySer (blue). Scale bar: 20 µm (B–E).</p

Co-expression of the β4 subunit reduces cell-surface trafficking of BK channels.

<p>(A) Membrane topology of the FAP-BKα and β4 proteins. The FAP tag is at the extracellular, N-terminus. The C-terminus of the BKα subunit is indicated. (B) Schematic of binding of cell-impermeable dye (pink) to the FAP results in significant increase in fluorescence (red). (C) Application of cell-impermeable dye labels only surface BKα channels in live HEK-293 cells co-transfected with FAP-BKα (red) and GFP (green). (D) Same as (C) but in cells co-transfected with β4, FAP-BKα, and GFP showing reduced surface expressed of the FAP-BKα. (E) Application of cell-permeable dye labels intracellular stores of channel in cells transfected with FAP-BKα (red) and GFP (green). (F) Same as (E) but in cells transfected with β4, FAP-BKα, and GFP. Scale bar = 20 µm (C–F). (G) Distribution of surface fluorescence intensity values after application of cell-impermeable dye in transfected cells for FAP-BKα (red bars) or FAP-BKα+β4 (black bars). (H–I) Proposed model for surface distribution of BK channels in the absence (H) or presence (I) of the β4 subunit.</p

β4-containing BK channels do not contribute to whole-cell BK channel currents.

<p>(A) In situ hybridization showing regions of β4 expression in a mouse brain. Arrow points to high levels of expression in the CA3. (B) Hippocampal area CA3 shows robust β4 expression. Scale bar = 200 µm. (C) Bright field image showing a whole cell electrode in the CA3. Scale bar = 500 µm. (D) Fluorescent image of a CA3 pyramidal cell filled with Alexa fluor 568 after whole cell patch clamp recording. Scale bar = 50 µm. (E) Overlaid traces from a representative cell showing total potassium current and the residual potassium current after application of a BK channel blocker. (F) Schematic overlap between paxilline- and iberiotoxin-sensitive currents when β4-containing BK channels contribute to whole-cell current. (G) Schematic overlap between paxilline- and iberiotoxin-sensitive currents when β4 containing BK channels do not contribute to whole-cell current. (H) Example paxilline-sensitive BK channel current from a representative cell. (I) Example iberiotoxin-sensitive BK channel current from a representative cell. (J) Quantitation of mean paxilline (pax)- and iberiotoxin (ibtx)-sensitive currents from CA3 pyramidal cells.</p