Diverse unassembled TMDs mediate ER localisation.

<p>(A) Cartoon schematic of the domain structure of CD8α. The TMD amino acid sequence and predicted ΔG_app for TMD membrane insertion of CD8^<i>WT</i>, CD8^<i>TMD*</i>, CD8^<i>PMCA</i> and CD8^<i>SERCA</i> are shown. (B) Cells expressing CD8^<i>WT</i>, CD8^<i>TMD*</i>, CD8^<i>PMCA</i> or CD8^<i>SERCA</i> were fixed with formaldehyde, permeabilised with Triton X-100 and co-immunostained with antibodies against BAP31 and the epitope tag HA. Cell nuclei were stained with DAPI (represented in blue in merged images). Scale bars indicate 10 μm.</p

CD8^<i>TMD*</i> retrieval from the Golgi is mediated by Rer1.

<p>(A) Helical wheel projections for the transmembrane domains of CD8^<i>TMD*</i> and CD8^<i>PMCA</i>. Numbering starts from the N-terminus of the predicted TMD. (B) Rer1 mRNA levels were determined by qPCR 72h post transfection with scrambled or Rer1 siRNA and normalised relative to GAPDH mRNA levels. (C and D) Cells expressing CD8^<i>TMD*</i> were transfected with scrambled siRNA or siRNA targeting Rer1 as indicated. 72 hours subsequently, cells were fixed and co-immunostained with antibodies against (C) ERGIC53 and HA or (D) EEA1 and HA. Cell nuclei were stained with DAPI. Scale bars indicate 10 μm. (E) Cell expressing CD8^<i>TMD*</i> were transfected with scrambled siRNA or siRNA targeting Rer1 for 72 hours. Immediately prior to harvesting, cells were incubated with leupeptin and pepstatin A (L/P) for 5 hours or left uninhibited. Whole cell lysates were separated by SDS-PAGE and analysed by immunoblotting with antibodies against HA. (F) Signal intensities from high molecular weight ‘i’ and ‘m’ glycoforms expressed as a ratio to the unprocessed ‘u’ glycoform. Scrambled siRNA vs RER1 siRNA + L/P, p = 0.0015; Rer1 siRNA vs Rer1 siRNA + L/P, p = 0.0267; scrambled siRNA + L/P vs Rer1 siRNA + L/P, p = 0.0337; one way ANOVA with Tukey’s multiple comparisons test. Data represents mean ±S.E.M. from 3 independent experiments.</p

Transmembrane domain quality control systems operate at the endoplasmic reticulum and Golgi apparatus

<div><p>Multiple protein quality control systems operate to ensure that misfolded proteins are efficiently cleared from the cell. While quality control systems that assess the folding status of soluble domains have been extensively studied, transmembrane domain (TMD) quality control mechanisms are poorly understood. Here, we have used chimeras based on the type I plasma membrane protein CD8 in which the endogenous TMD was substituted with transmembrane sequences derived from different polytopic membrane proteins as a mode to investigate the quality control of unassembled TMDs along the secretory pathway. We find that the three TMDs examined prevent trafficking of CD8 to the cell surface via potentially distinct mechanisms. CD8 containing two distinct non-native transmembrane sequences escape the ER and are subsequently retrieved from the Golgi, possibly via Rer1, leading to ER localisation at steady state. A third chimera, containing an altered transmembrane domain, was predominantly localised to the Golgi at steady state, indicating the existence of an additional quality control checkpoint that identifies non-native transmembrane domains that have escaped ER retention and retrieval. Preliminary experiments indicate that protein retained by quality control mechanisms at the Golgi are targeted to lysosomes for degradation.</p></div

CD8^<i>TMD23</i> is predominantly retained in the Golgi.

<p>(A) Whole cell lysates of cells expressing CD8^<i>WT</i>, CD8^<i>TMD*</i> or CD8^<i>TMD23</i> were separated by SDS-PAGE and analysed by immunoblotting with antibodies against HA. (B) Ratios of signal intensities of intermediate ‘i’ and mature ‘m’ to unprocessed ‘u’ glycoforms of CD8^<i>TMD*</i> or CD8^<i>TMD23</i> at steady state (n = 3). P = 0.0109, unpaired t-test. (C & D) Cells expressing (C) CD8^<i>TMD23</i> or (D) CD8^<i>WT</i> were pulse labelled with [³⁵S] met/cys for 10 minutes and chased for up to 90 minutes as indicated. Samples were immunoprecipitated with antibodies against HA, separated by SDS-PAGE and analysed by phosphorimaging. (E) In parallel, cells expressing CD8^<i>WT</i> or CD8^<i>TMD23</i> were chilled on ice and labelled with antibodies against the extracellular domain of CD8 prior to fixation in formaldehyde. Note that pictures were taken in parallel with equal exposure times. Scale bars indicate 10 μm. (F) Cells expressing CD8^<i>WT</i> or CD8^<i>TMD23</i> were chilled on iced water and labelled with biotin prior to cell lysis. Total cell lysates samples were taken and the remaining cell lysate incubated with neutravidin to isolate biotinylated (cell surface) protein. Total ‘T’ and biotinylated ‘B’ samples were separated by SDS-PAGE and analysed by immunoblotting with antibodies against HA, tubulin and Hsp70. (G) Cells expressing CD8^<i>WT</i> or CD8^<i>TMD23</i> were incubated on ice and treated with trypsin. Subsequently cells were washed and whole cell lysates separated by SDS-PAGE and analysed by immunoblotting with antibodies against HA and actin.</p

CD8^T<i>MD23</i> is degraded by proteasomes and lysosomes.

<p>(A) Cells expressing CD8^<i>WT</i> or CD8^<i>TMD23</i> were treated with cycloheximide (CHX) to prevent further protein synthesis. Whole cell lysates were harvested at the indicated chase time, separated by SDS-PAGE and analysed by immunoblotting with antibodies against HA and the loading control actin. Where indicated, cells expressing CD8^<i>TMD23</i> were treated with leupeptin and pepstatin A (LP) or PSII concurrently with CHX. (B) Signal intensities from (A) were quantified, normalised relative to the loading control and expressed as a percentage of the protein level at the start of the chase. Data represents mean ±S.E.M. from 3 independent experiments.</p

CD8^<i>TMD*</i> and CD8^<i>PMCA</i> are retrieved from the Golgi.

<p>(A) Cells expressing CD8^<i>TMD*</i> were pulse labelled with [³⁵S] met/cys for 10 minutes and chased for up to 90 minutes as indicated. Samples were immunoprecipitated with antibodies against HA, separated by SDS-PAGE and analysed by phosphorimaging. (B) Whole cell lysates of cells expressing CD8^<i>WT</i>, CD8^<i>TMD*</i> or CD8^<i>PMCA</i> were separated by SDS-PAGE and analysed by immunoblotting with antibodies against HA and tubulin. (C-E) Cells expressing (C) CD8^<i>TMD*</i>, (D) CD8^<i>PMCA</i> or (E) CD8^<i>WT</i> were incubated at 15°C or 37°C for 3 hours prior fixation. Cells were co-immunostained with antibodies against ERGIC53 and the epitope tag HA. Scale bars indicate 10 μm.</p

Transmembrane domain quality control at the ER and Golgi.

<p>A model for the handling of proteins containing misassembled transmembrane domains (red ovals). (1) Proteins containing misassembled transmembrane domains may be retained in the ER and degraded via ERAD. (2) A proportion of these proteins may cycle between the ER and Golgi. Golgi-to-ER retrieval is at least in part mediated by Rer1 (blue oval). (3) Proteins that are not retrieved to the ER may be retained by Golgi quality control machinery. (4) Proteins that escape ER retrieval are degraded in lysosomes. Some protein may traffic to the cell surface. It is not clear whether trafficking to the plasma membrane is a prerequisite for targeting to lysosomes, or whether proteins containing aberrant TMDs can be directly targeted to the endo/lysosomal system from the Golgi.</p

CD8^<i>TMD23</i> is a substrate from Golgi quality control.

<p>(A) Cartoon schematic of the domain structure of CD8α. The amino acid compositions and predicted ΔG_app for TMD membrane insertion of CD8^<i>TMD*</i> and CD8^<i>TMD23</i> are shown. (B) Helical wheel projection for the transmembrane domain of CD8^<i>TMD23</i>. Numbering starts from the N-terminus of the predicted TMD. (C and D) Cells expressing CD8^<i>TMD*</i> or CD8^<i>TMD23</i> were fixed and co-immunostained with antibodies against (C) BAP31 or (D) GM130 as well as the epitope tag HA in all cases. Cell nuclei were stained with DAPI. Scale bars indicate 10 μm.</p

Pipette–Surface Interaction: Current Enhancement and Intrinsic Force

There is an intrinsic repulsion between glass and cell surfaces that allows noninvasive scanning ion conductance microscopy (SICM) of cells and which must be overcome in order to form the gigaseals used for patch clamping investigations of ion channels. However, the interactions of surfaces in physiological solutions of electrolytes, including the presence of this repulsion, for example, do not obviously agree with the standard Derjaguin–Landau–Verwey–Overbeek (DLVO) colloid theory accurate at much lower salt concentrations. In this paper we investigate the interactions of glass nanopipettes in this high-salt regime with a variety of surfaces and propose a way to resolve DLVO theory with the results. We demonstrate the utility of this understanding to SICM by topographically mapping a live cell’s cytoskeleton. We also report an interesting effect whereby the ion current though a nanopipette can increase under certain conditions upon approaching an insulating surface, rather than decreasing as would be expected. We propose that this is due to electroosmotic flow separation, a high-salt electrokinetic effect. Overall these experiments yield key insights into the fundamental interactions that take place between surfaces in strong solutions of electrolytes

Image_2_Memory discrimination is promoted by the expression of the transcription repressor WT1 in the dentate gyrus.tif

The hippocampus is critical for the precise formation of contextual memories. Overlapping inputs coming from the entorhinal cortex are processed by the trisynaptic pathway to form distinct memories. Disruption in any step of the circuit flow can lead to a lack of memory precision, and to memory interference. We have identified the transcriptional repressor Wilm’s Tumor 1 (WT1) as an important regulator of synaptic plasticity involved in memory discrimination in the hippocampus. In male mice, using viral and transgenic approaches, we showed that WT1 deletion in granule cells of the dentate gyrus (DG) disrupts memory discrimination. With electrophysiological methods, we then identified changes in granule cells’ excitability and DG synaptic transmission indicating that WT1 knockdown in DG granule cells disrupts the inhibitory feedforward input from mossy fibers to CA3 by decreasing mIPSCs and shifting the normal excitatory/inhibitory (E/I) balance in the DG → CA3 circuit in favor of excitation. Finally, using a chemogenetic approach, we established a causal link between granule cell hyperexcitability and memory discrimination impairments. Our results suggest that WT1 enables a circuit-level computation that drives pattern discrimination behavior.</p