139 Matching Annotations
  1. Dec 2022
    1. transgene

      See transgenic above

    2. imaginary discs

      A group of undifferentiated cells that eventually form the drosophila wing.

    3. in vivo

      Experiments are carried out within the living organisms as opposed to the experiments in test tubes or Petri dishes. Up until here, all experiments done in this paper are in vitro.

  2. Nov 2022
    1. Xenopus laevis oocytes

      Cells of a type of frogs (Xenopus laevis) native to sub-Saharan Africa. These are used as model organisms for studying cell function such as ion channel which is reported in this paper

    2. Fig. 1. RECS1 induces cell death through the mitochondrial pathway of apoptosis. (A) Left: MEFs expressing doxycycline-inducible Flag-RECS1 were stimulated with doxycycline (DOX), and the levels of RECS1 were determined by Western blot. Right: Cell death was determined by PI and SYTO16 costaining followed by automated microscopy (n = 7). Mw, weight-average molecular weight; NT, not treated. (B) MEFs were transiently transfected with the indicated concentrations of MYC-RECS1 followed by Western blot analysis. (C) Left: Bright-field (BF) and PI fluorescent images of MEFs transfected with 0.4 μg of MYC-RECS1 for 24 hours. Right: Cell death was determined by PI staining followed by FACS (n = 3). (D) Left: Images of MEF Flag-RECS1 cells cultured with doxycycline in the presence or absence of the caspase inhibitor QvD-OPh (QvD; 40 μM) for 24 hours (red, PI staining). Right: Corresponding cell death kinetics (n = 3). (E) PI staining and FACS of cells treated as in (D) (n = 4). (F) MEF Flag-RECS1 wild-type (WT) or BAX and BAK (BB) double-knockout (DKO) cells were treated with doxycycline, and indicated proteins were assessed by Western blot. (G) Indicated cells were cultured with doxycycline for 38 hours, and cell death was determined by FACS (n = 3). (H) Images (left) and quantification (right) of RECS1 (Flag) colocalization with indicated organelle markers in Flag-RECS1 MEFs treated with doxycycline. GA, Golgi apparatus. Data represent means ± SD (A, C, and D) or means ± SEM. Statistically significant differences were determined comparing the best individual fit for each curve using the extra-sum-of-squares F test (A and D) or two-way analysis of variance (ANOVA) followed by Holm-Sidak’s multiple comparisons test (C, E, and G). *P < 0.05; **P < 0.01; ****P < 0.0001).


      Through which pathway does RECS1 induce cell death

      Western blot:

      A technique to analyze/detect the levels of a specific protein in the sample.

      Wild type (WT) Cells:

      These are normal cells with all expected qualities and properties.

      BAX and BAK (BB) double-knockout (DKO) cells: Cells in which both BAX and BAK have been artificially made non-functional.


      Doxycycline was used to control (induce) expression of RECS1. (left) RECS1 expression increased over 24 hours as measured by western blot using anti-FLAG antibody. (Right) Induction of expression of RECS1 by doxycycline alone caused about 20% increase in cell death after 28 hours. Cell death was estimated from the number of cells with propidium iodide stain.


      Increased concentration of plasmid result in more RECS1 in cells. Cells were transformed with MYC-RECS1, and the level of this protein was detected by western blot.


      Cell death increased at higher concentration (overexpression) of RECS1. Here, MYC-RECS1 plasmid was introduced into cells and then treated with PI. Cells that stain red are dead and are more when 0.4ug of MYC-RECS1 is applied, compared to when 0.1ug is applied.

      1D & E.

      QvD-OPh (QvD) is an inhibitor of BAX and BAK proteins. These are caspases that mediate cell death through the mitochondria. Inhibiting these proteins reduced apoptosis.


      Overexpressing RECS1 in cells lacking BAX and BAK prevents cell death. Therefore, RECS1 induces cell death through BAK and BAX i.e., though the mitochondrial apoptosis pathway.


      RECS1 colocalizes with lysosome markers LAMP1, LAMP2 and GM130 but not the ER marker, ERp72. Here, it regulates the susceptibility of cells to lysosomal stress.

    3. Apoptosis

      A programmed and normal process of cell death. See the YouTube videos here for a little more detail https://www.youtube.com/watch?v=DR80Huxp4y8 https://www.youtube.com/watch?v=F4lUnOY0U5w

    4. transmembrane domains

      Certain sections are located across (intersect) membranes such as cell membrane, lysosome membrane, mitochondria membrane etc.

    5. Fig. 9. RECS1-like/CG9722 modulates viability in response to intracellular stress in D. melanogaster. (A) One copy of dRECS1 was overexpressed in the wing imaginal discs of animals fed with control food or food supplemented with CQ (100 μg/ml) or Tm (10 μg/ml). The phenotype of adult male (right) and female (left) wings was quantified (n = 3 to 7, >80 wings per condition). (B) Control (yw) and mutant (dRECS1KO) male and female flies were treated with Tm (10 μg/ml), and the animal death rate was quantified daily (n = 2 to 3). (C) WT, dRECS1KO, or dRECS1IR pupae were treated with Tm, and the percentage of eclosion was quantified (n = 2 to 4). (D) WT and dRECS1KO female flies were fed a normal diet or starved, and the death rate was daily quantified (n = 3). (E) WT and dRECS1KO knockout male flies were fed a normal diet or starved, and the death rate was daily quantified (n = 2 to 3). Data represent means ± SEM. Statistically significant differences were determined by two-way ANOVA followed by Holm-Sidak’s multiple comparisons test. ****P < 0.0001.

      Figure 9


      In the presence of lysosomal stress inducers, TM and CQ, overexpression of dRECS1 amplified abnormal wing development in flies.

      9B & C

      Flies with reduced dRECS1 expression exhibit longevity in ER stress-inducing conditions

      9D & E

      dRECS1KO female flies (flies without RECS1 expression) are more resistant to nutrient starvation. CG9722 (dRECS1) regulates ER and lysosomal stress-induced apoptosis in flies

    6. Fig. 7. RECS1 is a pH-dependent calcium and sodium channel. (A) Alignment showing the conservation of the critical Arg60 residue from BsYetJ. Right: Comparison of the conserved di-aspartyl sensor. (B) Tridimensional structural model of the human RECS1 in closed and open conformations. Zoom: Key conserved C-terminal residues involved in pH sensing and channel opening. (C) X. laevis oocytes were injected with mRNA for Flag-RECS1 WT or D295Q, and ionic single-channel currents were recorded in the cell-attached mode using patch clamp at pH 7 or 6.5. Inset: Amplified plots depicting three opening states for RECS1 (O1, O2, and O3). (D) Quantification of the probability to find the channel in the open state (NPo) for RECS1 WT and D295Q (left). The NPo for open states O1 (middle) and O2 (right) is also shown [n = 4 (RECS1 WT) or n = 3 (RECS1 D295Q)]. (E) Images of X. laevis oocytes injected with different concentrations of Flag-RECS1 (top) and oocyte survival (bottom). Arrowhead indicates morphological alterations (n = 3). NI, not injected. (F) Recordings of macroscopic currents using the cut-open technique. (G) Permeability ratios with respect to K+ for indicated cations (n = 3 to 7). (H) Top: Indicated MEF Flag-RECS1 cells were treated with doxycycline, and protein levels were monitored by Western blot. Dashed lines indicate Western blot splicing. Bottom: Cells were treated with doxycycline and 25 μM CQ for 24 hours. Cell death was determined by FACS and normalized to WT (100%) (n = 3). Data represent means ± SEM. Statistical differences were determined by two-way (D, E, and G) or one-way ANOVA followed by Holm-Sidak’s multiple comparisons test. *P < 0.05; ***P < 0.001; ****P < 0.0001.

      Figure 7

      Driving question: What is the possible biochemical role or nature of RECS1?

      7A & B

      Multiple sequence alignment of proteins related to RECS1, based on a critically conserved Arg 60 residue of a closely related bacteria BsYetJ (7A). MODELLER platform was used to construct a 3-D structural model of RECS1 in both the open and closed states. Model suggests RECS1 to be an ion channel, particularly Asp 295 as a critical residue in this function (senses pH changes and regulates opening). An equivalent Asp residue is critical in other related proteins.

      7C & D

      Testing the electrophysiological properties of RECS1. Does RECS1 allow ionic movements? Under what conditions? At a neutral pH, 7 and constant voltage, both RECS1 WT and mutant cells exhibited similar spontaneous current spikes, which reduced at lower pH (6.5) in three open states (01-03), suggesting that RECS1 is pH-dependent channel. Probability of finding RECS1 in an open state also reduces with pH, confirming that RECS1 activity is pH-dependent.


      Frog larvae (Oocytes) overexpressing RECS1 die in RECS1-dose dependent manner, consistent with the role of RECS1 a death regulator.

      7F & G

      Ca2+ ions are more permeable that Na+ ions in RECS1-dependent manner (F), while mutation of RECS1 at Asp295 hinders influx of Ca2+ and Na+ but not Ba+ or Cs2+ (G).


      Overexpression of D295Q (RECS1 mutant) blocks lysosomal stress induced MEF cell death, thus functional RECS1 is required for lysosomal stress-induced cell death regulation.

    7. Fig. 5. Inhibition of the endogenous RECS1 protects cells against ER stress. (A) Knockdown of the endogenous mouse RECS1 using two different siRNA pools was confirmed by quantitative polymerase chain reaction (qPCR). (B) Left: Cells transfected with RECS1 siRNAs were treated with Tm (50 ng/ml), and cell death was determined by SYTOX green staining. A representative experiment from four independent experiments is shown. Right: Quantification of cell death at 24 hours (n = 4). (C) Cell death kinetics of cells transfected as in (B) and treated with 25 nM Tg. Left: Representative kinetics. Right: Quantification at 24 hours (n = 3). Data are shown as means ± SD. Statistical differences were determined by one-way ANOVA followed by Holm-Sidak’s multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001.

      What is the function of native RECS1 in cell death regulation and control of ER stress.


      RNA interference was used to transiently reduce expression of RECS1 in MEFs. No effect on basal cell viability was observed.

      5B & C

      Silencing RECS1 reduced the effect of Tm and Tg- induced ER stress on MEF cells.

    8. Fig. 4. RECS1 sensitizes cells to ER stress. (A) Annexin V versus PI FACS density plots from MEF Flag-RECS1 cells cultured with doxycycline for 16 hours and then treated with Tm (100 ng/ml) for 16 hours followed by FACS (n = 9). (B) Experimental design for cell death kinetic experiments. (C) Cell death kinetic analyses of MEFs treated as in (A) (n = 6). (D) Cell death of MEF Flag-RECS1 cells incubated with doxycycline and then treated with 100 nM Tg for 24 hours (n = 3). (E) Corresponding cell death kinetic analysis (n = 3). (F) Clonal assay of MEF Flag-RECS1 cells treated with doxycycline alone or in the presence of Tm (50 ng/ml) or 25 nM Tg. (G) Cell death kinetics of MEF Flag-RECS1 cells cultured with doxycycline and then treated with Tm (100 ng/ml) in the presence or absence of 40 μM QvD (n = 3). (H) MEF Flag-RECS1 WT and BAX and BAK DKO cells were cultured with doxycycline and treated with Tm (100 ng/ml) or 50 nM Tg for 24 hours. Cell death was determined by FACS (n = 2 to 3). Data are shown as means ± SD (C, E, and G) or means ± SEM. Statistical differences were determined by the extra-sum-of-squares F test (C, E, and G) or two-way ANOVA followed by Holm-Sidak’s multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

      What is the effect of RECS1 over expression on ER- induced cell death


      Overexpression of RECS1 in ER stressed cells (TM treatment) triggers cell death. Dead cells are shown by annexin V and PI.

      4B & C

      Cell death correlates with ER stress induction and progression in RECS1 over expressing MEF cells

      4D & E

      Cell death correlates with ER stress induction and progression in RECS1 over expressing cells treated with calcium pump inhibitor, Thapsigargin (Tg).


      Cells over expressing RECS1 have reduced long term survival and reduced growth under ER stress

      4G & H

      QvD and Tm (G), BAX and BAK deficiency inhibited RECS1-triggered cell death under ER stress as expected.

    9. Fig. 3. RECS1 causes LMP in cells undergoing stress. (A) HeLa Flag-RECS1 cells were cultured with doxycycline and then treated with indicated concentrations of CQ, QvD, or both for 16 hours. Immunofluorescence against galectin-1 was performed. (B) Quantification of galectin-1 puncta per field (n = 2; 6 to 27 fields in total). (C) MEF Flag-RECS1 cells were cultured with doxycycline and treated with 50 μM CQ for indicated time points. The enzymatic activities of cathepsin D, acid phosphatase, and hexosaminidase were assessed from cytosolic, lysosomal-free fractions (n = 3). (D) MEF Flag-RECS1 WT and BAX and BAK DKO cells were cultured with doxycycline and then treated with indicated concentrations of CQ for 24 hours. Cell death was determined by PI staining followed by FACS (n = 3). (E) MEF Flag-RECS1 cells were cultured with doxycycline and treated with CQ for 16 hours. Immunofluorescence against LAMP-2 (green), BAX N-terminal domain (red, BAX 6A7 antibody), and Flag-RECS1 (blue, anti-Flag antibody) was performed. The triple colocalization of Flag-RECS1, LAMP-2, and active BAX is visualized as white color. (F) Representative immunofluorescence images of Flag-RECS1 MEF cells treated as in (E). Bottom panels correspond to zoomed white boxes (marked as z) from the top panels. (G) Intensity profiles for LAMP-2, BAX, and Flag-RECS1 signals along the line shown with an L1 (left) or an L2 (right) in (F). Data are shown as means ± SEM. Statistically significant differences were determined by two-way ANOVA followed by Holm-Sidak’s multiple comparisons test. **P < 0.01; ***P < 0.001; ****P < 0.0001.

      Effect of RECS1 expression on Lysosome membrane permeabilization

      3A & B

      After LMP, the cytosolic proteins LGALS1/galectin-1 and LGALS3/galectin 3 relocate to the lysosomal glycocalyx and form punctate structures. Cells overexpressing RECS1 had increased LGALS1/galectin-1 puncta at high chloroquine doses.


      The activity of lysosome enzymes, notably cathepsin D, acid phosphatase and hexosaminidase is increased in RECS1 overexpressing cells under chloroquine treatment.


      The resistance of BAK and BAX DKO cells to lysosomal stress-induced cell death confirms that these caspases are required in this process.

      3E & G

      BAX colocalizes with RECS1 in in LAMP2 containing vesicles and redistributes to larger LAMP2 puncta, specifically in RECS1-positive lysosomes upon CQ treatment in a RECS1-dpemdndent manner.

    10. Fig. 2. RECS1 triggers cell death upon lysosomal stress. (A) Flag-RECS1 MEFs were stimulated with doxycycline for 16 hours and treated with a library of 77 cytotoxic compounds for 24 hours. Cell death was determined by PI staining using an ImageXpress Micro XL automated microscope. ETO, etoposide; NVB, vinorelbine tartrate; DTX, docetaxel. (B) Heatmap of the top 10 sensitizing compounds in (A). For each compound, heatmap colors depict doxycycline-treated PI values after the subtraction of cell death induced by doxycycline alone and by the compound in the absence of doxycycline. *CQ and HCQ were used at 50 μM. **Cisplatin (CDDP) was used at 150 μM. (C) Left: Representative images of MEF Flag-RECS1 cells pretreated with doxycycline and treated with 50 μM CQ for 24 hours (red, PI staining). Right: Cell death kinetic analyses of cells treated as in (B) (n = 3). (D) Cells were cultured as in (C) and then treated with indicated concentrations of bafilomycin A1 for 24 hours. Cell death was determined by FACS (n = 4). (E) Left: Representative images of HeLa Flag-RECS1 cells treated as in (C). Right: Corresponding cell death kinetic analyses (n = 3). (F) HeLa Flag-RECS1 cells were treated as in (E), and cell death was assessed by FACS (n = 3). Data are shown as means ± SD (C and E) or means ± SEM (A, D, and F). Statistically significant differences were determined using the extra-sum-of-squares F test (C and E) or two-way ANOVA followed by Holm-Sidak’s multiple comparisons test. *P < 0.05; **P < 0.01; ****P < 0.0001.

      Role of RECS1 in stress-induced cell death

      2A & B

      Cell death was triggered in RECS1-overexpressing cells treated with a subset of lysosomal stress-inducing drugs but not other drugs. Chloroquine and Hydroxychloroquine treatments were associated with the highest cell death

      2C & D

      Treatment of RECS1 overexpressing MEF cells with Chloroquine (lysosomal stressor) or Vacuolar ATPase inhibitor, bafilomycin resulted in over 80% cell death in 24 hours, suggesting that RESC1 mediates cell death triggered by lysosomal stress.

      2E & F

      Treatment of RECS1 overexpressing Hela cells with Chloroquine or bafilomycin resulted in over 60% cell death in 24 hours.

    11. anterior-posterior shortening

      Reduced body length

    12. We found that dRECS1KO cells exhibited increased formation of autophagosomes and autolysosomes together with higher overall autophagy flux (fig. S8, D to F). Together, these results suggest that CG9722 has a proapoptotic activity in vivo and sensitizes flies to lysosomal- and ER stress–induced cell death.

      Flies without RECS1 exhibit increased autophagy under stress

    13. autophagy

      Self eating by cells, that is break down of cell components to recycle nutrients for use by the cells

    14. In addition, dRECS1KO female animals were more resistant to death induced by nutrient starvation, an effect that was still observed but not as prominent in male flies or larvae (Fig. 9, D and E).

      Female flies without RECS1 also exhibit increased lifespan even when starved by withholding food.

    15. Moreover, dRECS1 deficiency conferred resistance to Tm in male and female flies, increasing life span under ER stress (Fig. 9, B and C).

      Male flies without RECS1 have longer lifespan, even when they were stressed with Tm

    16. wing phenotype

      External appearance/morphology of wings

    17. transposon

      Jumping genes/DNA

    18. Last, to define the relationship between the channel activity of RECS1 and the regulation of cell death induced under lysosomal stress, we generated a doxycycline-inducible MEF cell line expressing a single-point mutant version of RECS1 (Fig. 7H). When compared to WT RECS1, the overexpression of the D295Q mutant diminished cell death, behaving similarly to the mock conditions (Fig. 7H)

      As opposed to normal RECS1, a high dose of non-functional RECS1 (D295Q) has no effect on cell viability/cell death, suggesting that the function of RECS1 as ion channel is linked to its role in cell death regulation

    19. Mutation of the di-aspartyl sensor results in a marked decrease in the channel’s permeability to Ca2+ and Na+ but not to Ba2+ or Cs+ (Fig. 7G). Together, these results suggest that RECS1 is a pH-dependent cationic channel, with a main preference for calcium.

      Reduced calcium entry is observed when RECS1 is non functional

    20. ltage clamp technique with potassium as the reference ion. Analyses of permeability ratios indicated that RECS1 allows the flux of Ca2+ and, to a lesser extent, Na+ (Fig. 7F).

      RECS1 allows in more Calcium than Sodium

    21. Notably, overexpression of RECS1 triggered cell death and structural abnormalities in the oocytes in a dose-dependent manner, consistent with its role as a cell death regulator (Fig. 7E)

      Increasing the dose of RECS1 amplified cell death in oocytes.

    22. Mutation of the key Asp295 from the putative di-aspartyl sensor significantly reduced RECS1 opening probability at pH 7, while it only slightly affected the ability to respond to pH changes (Fig. 7D).

      Asp295 is required as a pH sensor, because when cells with non-functional Asp295 are used, the probability of finding the channel in open state is significantly reduced at pH 7.

    23. Lowering the pH to 6.5 caused a marked decrease in the number of current spikes in both channels (Fig. 7C), consistent with the hypothesis of RECS1 as a pH-regulated channel. Quantification of the total open probability (NPo) in addition to the probability for each independent opening state (NPo Ox) showed that the channel activity of RECS1 is heavily dependent on pH (Fig. 7D).

      Lower amount of electricity is conducted at lower pH, suggesting that the ion channel function is regulated by pH.

    24. current spikes

      Indication of electrical current strength

    25. single-channel currents in the cell-attached mod

      Electrical current through the channel is proportional to the amount of ions going through the channel.

    26. electrophysiological properties

      Electrical properties of cells. These are related to the role of RECS1 as an Ion channel

    27. MODELLER platform

      A web-based computer program for predicting the 3D protein structures. Check it out at the link below. https://www.salilab.org/modeller/

    28. interaction between Arg158 with the Asp268-Asp295 di-aspartyl sensor in the closed conformation (Fig. 7B, green)

      The interaction of Arg158 with the Asp268-Asp295 maintains a closed state, leading to regulation of ion movements

    29. In the open state, this latch becomes dislodged, and the second transmembrane is displaced out, leading to the opening of the channel (Fig. 7B, magenta). Our model suggests that RECS1, as demonstrated for other proteins of the LFG and BI-1 families (11, 12, 25), could operate as an ion channel. Our bioinformatic analysis also suggests that Asp295 (D295) is a strong candidate to regulate the channel activity of RECS1 and was therefore selected for mutagenesis and functional studies.

      The authors think that Asp295 is very important in regulating the activity of this predicted ion channel. They then make this residue non-functional to study its function in cell death

    30. homology models of the three-dimensional (3D)

      Models based on closely related proteins of known structures.

    31. morphology

      Structure, shape, size

    32. Fig. 6. RECS1 increases lysosomal acidification and calcium content. (A) Lysosomal pH of HeLa Flag-RECS1 loaded with FuraDx and OGDx and cultured in the absence or presence of doxycycline for 24 hours (n = 5, 15 to 16 total coverslips). (B) Corresponding lysosomal calcium concentration. (C) Left: Cytosolic calcium levels were determined in HeLa Flag-RECS1 cells treated with doxycycline. Cells were treated with 5 μM Tg to stimulate ER calcium release. Right: Maximum ER calcium peak (n = 4, 10 to 11 coverslips). ns, not significant. (D) MEF WT and BAX and BAK DKO Flag-RECS1 cells were loaded as in (A), and lysosomal pH was determined after 24 hours of doxycycline treatment (n = 6, 12 to 17 coverslips per condition). (E) Lysosomal calcium measurements (n = 6). (F) Knockdown of the endogenous mouse RECS1 by siRNA was confirmed by qPCR after 48 hours of transient transfection. (G) Cells transfected as described in (F) were loaded as described in (A), and lysosomal pH was determined after 48 hours of transfection (n = 5, 14 to 15 coverslips). (H) Lysosomal calcium measurements (n = 5, 14 to 15 coverslips). (I) MEF Flag-RECS1 WT and BAX and BAK DKO cells were treated with doxycycline for 16 hours and analyzed by TEM. Image magnification, ×4000 (n = 13 to 17 cells per condition). (J) Diameter of primary and secondary lysosomes (n = 8 to 13 cells per condition). Data represent means ± SEM. Statistically significant differences were detected using ratio-paired two-tailed t test (A, B, D, E, G, and H) or two-way ANOVA followed by Holm-Sidak’s multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

      Role of RECS1 in lysosomal pH regulation and calcium accumulation.

      6A, B & C

      Lysosomal pH was reduced in RECS1 overexpressing HeLa cells (A), while calcium concentration increased about 2-fold under these conditions (B), but no effect on ER calcium concentration (C).

      6D & E

      The reduction in lysosomal pH in MEFs is dependent on BAX and BAK expression and a over three-fold increase in calcium content was observed in cells overexpressing RECS1.

      6F, G & H

      RECS1 silenced cells have increased lysosomal pH (F, G), whereas the luminal pH was only slightly reduced when RECS1 was inhibited (H).

      6 I & J

      Number of secondary lysosomes but not size increased in cells over expressing RECS1 in BAX/BAK expression-dependent manner, suggesting an increased catabolic.

    33. ependent on BAX and BAK expression (Fig. 6D)

      RECS1 requires BAX and BAK to induce lysosomal pH and calcium level changes.

    34. RECS1 overexpression resulted in lysosomal acidification in HeLa cells, with lysosomal pH (pHL) decreasing from 4.77 to 4.38 (Fig. 6A). The acidification of lysosomes correlated with an increase in lysosomal intraluminal calcium concentration ([Ca2+]lys) from an average of 149 to 286 μM (1.9-fold increase) (Fig. 6B),

      Overexpression of RECS1 in cells is associated with reduced lysosomal pH and increased calcium in the lysosome.

    35. pH (OGDx)

      Fluorescence probe used for measuring lysosomal pH. Find more information about this probe at the following link: https://journals.biologists.com/jcs/article/115/3/599/34970/pH-dependent-regulation-of-lysosomal-calcium-in

    36. atiometric calcium (Fura-2Dx)

      Fura-2 is a ratiometric fluorescent dye which binds free intracellular calcium. Find more information about this dye at the following link: https://www.aatbio.com/resources/assaywise/2015-4-1/ratiometric-calcium-indicators

    37. calcium-binding affinities

      A measure of the readiness/ease by which a protein attaches/bind to calcium

    38. short hairpin RNA (shRNA)

      an artificial RNA molecule with a tight hairpin turn that can be used to silence target gene expression via RNA interference. Check out the following page for more information. https://horizondiscovery.com/en/applications/rnai/shrna-applications

    39. Silencing

      To reduce the expression of a specific gene, without completely stopping its function

    40. had no effects in basal cell viability (fig. S5, O and P), but it conferred protection against ER stress induced by Tm and Tg (Fig. 5, B and C).

      Reduced RECS1 expression blocks cell death even when ER stress is induced by Tm or Tg, suggesting that RECS1 induces cell death under ER stress

    41. (siRecs1#1 and siRecs1#2)

      Two siRNA would degrade/break down RECS1 and prevent its expression

    42. siRNAs

      Short RNA sequences that interfere with the expression of specific genes with sequences similar to the siRNA. The siRNA can be introduced into cells by transformation using plasmids. The siRNAs degrade specific mRNAs of target genes thereby preventing their translation into proteins. Here is a video about siRNA by Nature: https://www.youtube.com/embed/cK-OGB1_ELE?wmode=transparent

    43. endogenous RECS1

      RECS1 that naturally exist inside the cells

    44. (Fig. 4A). Since ER stress affects protein trafficking through the secretory pathway and the expression of RECS1 alone does not trigger ER stress, we allowed RECS1 to accumulate for 16 hours before challenging cells with Tm or other ER stressors (Fig. 4B). Using this protocol, we found that RECS1 overexpression hastened cell death to ER stress, as evidenced in cell death kinetic experiments (Fig. 4C). Moreover, we confirmed these observations in HeLa cells in dose-response experiments (fig. S5, M and N). We further validated these results in cells stimulated with Tg, followed by cell death measurements using both FACS and live microscopy (Fig. 4, D and E). Long-term survival assessment by clonogenic assays indicated that RECS1 expression reduced the growth of cells under ER stress (Fig. 4F). Last, we treated MEFs expressing Flag-RECS1 with QvD together with Tm and determined cell death by live microscopy. As expected, QvD treatment reduced cell death under ER stress (Fig. 4G). Similarly, BAX and BAK double deficiency fully inhibited cell death triggered by RECS1 overexpression under ER stress (Fig. 4H).

      Increased cell death is observed in cells overexpressing RECS1 and treated with ER stress inducers, Tm and Tg, suggesting that RECS1 induces ER-stressed cells to die.

    45. These results indicate that RECS1 overexpression alone does not cause ER stress. We also determined whether CQ treatment could induce ER stress. Treatment of Flag-RECS1–overexpressing MEFs with CQ failed to induce Xbp1 mRNA splicing (fig. S5, K and L), Bip/Grp78, or Chop/Gadd153 (fig. S5L). These results suggest that cell death induced by RECS1 under lysosomal stress conditions is not associated with the activation of the UPR.

      Neither overexpression of RECS1 a lone nor treatment with CQ induce UPR. Therefore, ER stress caused by Tm and other ER-stress inducing chemicals is not connected to UPR (does not cause UPR)

    46. mobility shift

      A technique used in biology to detect proteins bound to DNA based on their rate of movement in a gel. DNA-free protein appear smaller that the same protein bound to DNA.

    47. ip/Grp78 and Chop/Gadd153 (fig. S5H), two sensitive markers of ER stress (13, 15).

      Proteins whose expression changes during ER stress.

    48. UPR

      Unfolded protein response characterized by reduced protein production, or increased protein folding or increased protein degradation in response to ER stress.

    49. XBP1s

      X-box binding protein is a protein which accumulates in cells experiencing ER stress.

    50. Several proteins of the TMBIM family, such as BI-1 and GRINA, inhibit apoptosis triggered by ER stress, probably through the modulation of ER calcium (10, 13, 15, 17, 38). To gain further insights into the role of RECS1 in the control of cell death under cellular stress, we measured its expression in cells undergoing ER stress induced by the treatment with the N-glycosylation inhibitor tunicamycin (Tm). We found that Recs1 mRNA levels were up-regulated under ER stress, whereas Bi-1 mRNA levels remained unaltered (fig. S5A). This expression profile parallels that of certain BCL-2 proteins known to be induced by ER stress (i.e., Puma), while antiapoptotic components were unchanged (i.e., Bcl-2) (fig. S5B). Recs1 mRNA was also up-regulated in cells undergoing ER stress induced by the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) calcium pump inhibitor thapsigargin (Tg) (fig. S5C). The kinetics of the up-regulation of Recs1 mRNA under ER stress paralleled the up-regulation of the ER chaperone Bip/Grp78 and the proapoptotic factor Chop/Gadd153 (fig. S5D)

      Recs1 expression (mRNA) is increased in cells experiencing Endoplasmic Reticulum (ER) stress. Compounds that cause ER stress include tunicamycin (Tm), SERCA and thapsigargin (Tg)

    51. As expected, BAX and BAK DKO cells were almost completely resistant to cell death induced by treatment with 25 μM CQ (Fig. 3D). However, we observed 30% of cell death in BAX and BAK DKO Flag-RECS1 cells treated with the higher CQ dose (50 μM), an effect that could not be reversed by inhibition of mitochondrial transition permeability pore (mPTP) (cyclosporin A) or necroptosis (necrostatin-1s) (Fig. 3D and fig. S4G).

      Without BAX and BAK in cells, RECS1 cannot induce cell death even under stress conditions.

    52. In addition, we measured the enzymatic activities of three lysosomal enzymes—cathepsin D, acid phosphatase, and hexosaminidase—in cytosolic lysosomal-free fractions. In line with previous results, we found that the activity of these three enzymes is increased in the cytosol of MEFs expressing Flag-RECS1 after CQ treatment but not in control cells (Fig. 3C)

      Cells stressed with CQ and with high dose of RECS1 have more lysosomal protein leakage to the cytoplasm, suggesting that RECS1 induces LMP under lysosomal stress

    53. downstream

      A step in cellular pathway that occurs after another (upstream) step

    54. , treatment with CQ resulted in a dose-dependent increase in the number of LGALS1/galectin-1 puncta (Fig. 3, A and B

      Cells overexpressing RECS1 and stressed with CQ resulted in increased leak of proteins, suggesting that LMP was initiated

    55. LMP

      Lysosomal Membrane Permeabilization - When the lysosomal membrane is forced to take in substances which may otherwise be prevented from entry.

    56. During LMP, lysosomal intraluminal cathepsins and hydrolases leak into the cytosol, where they act as cell death proteases

      LMP results in the escape of protein-degrading proteins from the lysosomes to the cytoplasm, leading to initiation of cell death

    57. pathophysiological processes

      Abnormal changes in the body that result from disease or malfunctioning of the cells/body parts

    58. We then explored the effects of lysosomal stress on RECS1 localization. Analysis of the distribution of endogenous mouse RECS1 indicated that, under basal conditions, RECS1 colocalizes mainly in lysosomes and late endosomes (around ~20%) (fig. S3, C to E). However, in cells undergoing starvation or starvation and treated with CQ, RECS1 was further recruited to lysosomes but not to late endosomes, suggesting that RECS1 distribution is highly dynamic (fig. S3, C to E). Notably, the levels of Recs1 mRNA were up-regulated in cells undergoing nutrient starvation (fig. S3F). In addition, RECS1 overexpression induced autophagosome formation, an effect that was increased by CQ treatment (fig. S3G). Overall, our results indicate that RECS1 is located at lysosomes and regulates the susceptibility of cells to lysosomal stress.

      RECS1 is localized at the lysosomal membrane where it regulates cellular responses to stress conditions. It increases chances of cell death in these conditions.

    59. endogenous


    60. HeLa

      Another type of immortal cells used in biomedical research. For a primer on the origin and significancy of HeLa cells, see the following link. https://www.hopkinsmedicine.org/henriettalacks/

    61. colocalization analysis

      A technique to detect the location of one protein by use of the location of another protein. If two proteins exist in the same location together, then they are colocalized. Here, FLAG-RECS1 colocalizes with lysosomal proteins LAMP1 and LAMP2, suggesting that RECS1 is associated with the lysosome

    62. confocal microscopy

      Microscopy used to block most of the irrelevant light to produce good quality images. See a glimpse of the operating principle of a confocal microscope: https://en.wikipedia.org/wiki/File:Fluorescent_and_confocal_microscopes.ogv

    63. immunofluorescence

      A technique for determining the location of an antigen (or antibody) in tissues by reaction with an antibody (or antigen) labeled with a fluorescent dye.

    64. These results indicate that RECS1 potentiates cell death triggered by lysosomal stress.

      Altering lysosomal pH leads to stress. RECS1 causes rapid cell death under these conditions.

    65. bafilomycin A1

      Blocks lysosomal pH reduction by V-ATPase protein.

    66. V-ATPase (vacuolar H+-ATPase)

      Protein that lowers the pH of vacuoles/lysosomes.

    67. pH alkalization

      Increased PH

    68. lysosomotropic agents

      Compounds which induce lysosomal stress such as CQ and HCQ

    69. nexpectedly, RECS1 overexpression sensitized cells to the death triggered by ~14 compounds, whereas it repressed the activity of 12 compounds (fig. S2A). The top sensitizing drugs were chloroquine (CQ) and hydroxychloroquine (HCQ), which are known to induce lysosomal stress by inhibiting their acidification (Fig. 2, A and B).

      Up to 14 chemicals induced cell death in cells with high doses of RECS1 compared to cells with low RECS1. Chloroquine (CQ) and Hydroxychloroquine(HCQ), which are known to cause lysosome stress had more significant effect, suggesting that the observed death was related to lysosome function/stress.

    70. BAX- and BAK-dependent manne

      These are required for RECS1 to cause cell death

    71. depolarize

      Change the charge (negative or positive to neutral)

    72. Specific structural and functional criteria are required to validate a BH3 motif, since definitive bioinformatic procedures that rely purely on sequence analyses are currently lacking (28). To this end, we synthesized two peptides: RECS1280–299 and RECS1296–311, based on the N- and C-terminal BH3-like motifs, respectively.

      Two synthetic (non-natural) amino acid sequences were made . These amino acid sequences resemble the BH3-like motifs. These helped in determining the structure and function of RECS1

    73. deletion mutants: RECS1Δ910, lacking the C-terminal BH3 motif, and RECS1Δ868, lacking both

      Sequences of RECS1 with some nucleotides at the C-terminal removed. These targeted the two BH3-like segments.

    74. RECS1 has been traditionally classified within the TMBIM superfamily (8). However, detailed sequence and phylogenetic analyses suggest that RECS1, together with GRINA, LFG, GAAP, and Tmbim1b, forms a distinct and evolutionarily conserved protein family, termed the “LFG family,” characterized by a shared common ancestry and the lack of the signature BI-1 motif (fig. S1A) (7, 26). Previous sequence comparison using the BH3 motif consensus from Prosite (PS01259) identified one putative BH3 motif on the C-terminal domain of the human RECS1 and LFG proteins (22). The BH3-like motif in the C-terminal region of RECS1 contains the characteristic minimal Leu-X3-Gly-Asp/Glu sequence, but no additional BH3-like motifs of this kind were found in the other members of the LFG and BI-1 protein families (fig. S1B). However, an additional conserved BH3-like sequence consisting of Leu-X4-Asp is also present in an adjacent sequence of RECS1, in addition to GRINA and LFG (fig. S1B). These putative “BH3 motifs” are conserved in humans and in several species of non-human primates (fig. S1, C and D). However, only one such motif is present in rodents, suggesting that they represent a late evolutionary event.

      RECS1 sequence contains a short segment on the C-terminal that is thought to be involved in positive regulation of cell death. This short sequence is also found in other related proteins in other organisms.

    75. BAX and BAK double-knockout (DKO) background (Fig. 1F)

      Cells in which both BAX and BAK are non-functional

    76. caspases

      Proteins that degrade/breakdown other proteins (proteolysis). BAX and BAK are some of these caspases.

    77. o determine the requirement of caspases, we treated RECS1-overexpressing cells with the pan-caspase inhibitor QvD-OPh (QvD), followed by cell death kinetics and fluorescence-activated cell sorting (FACS) analysis. Caspase inhibition completely abrogated cell death induced by RECS1

      Without BAX and BAK, cell death was inhibited even when RECS1 was overexpressed, suggesting that to promote cell death, RECS1requires BAX and BAK function.

    78. intracellular signals

      Signals from within the cell

    79. These observations suggest that RECS1 overexpression is sufficient to induce cell death.

      RECS1 alone without any other treatment to cells induces cell death

    80. transient transfection

      Temporary expression of foreign plasmid DNA in cells. The foreign DNA does not get integrated into the cell genome.

    81. propidium iodide (PI)

      A red fluorescent dye that is used to stain DNA and RNA in dead cells. Only cells with a compromised membrane (dead cells) can allow it to enter. So, PI can be used as an indicator of the proportion of cells that are alive or dead.

    82. FLAG

      A small protein (peptide sequence) that is added to the end of the protein. In this case, FLAG was tagged onto RECS1 to enable detection of RECS1 in cells. Tagging is done through transformation

    83. doxycycline-inducible

      Expressed only in the presence of doxycycline antibiotic

    84. embryonic fibroblast (MEF)

      Fibroblast cells (cells that make connective tissue) from mouse embryos. These cells are immortalized (they grow indefinitely when maintained on specific media) and are used for research purposes.

    85. (D295Q)

      Aspartic acid (D) at position 295 in RECS1 is substituted for Glutamine (Q). This amino acid is necessary for regulation of lysosome membrane properties.

    86. proapoptotic

      Activates or positively regulates cell death

    87. mutation

      Change in amino acid or DNA sequence, resulting in altered function/structure of a protein

    88. LMP,

      Lysosomal Membrane Permeabilization - Making the lysosome membrane able to take up substance easily.

    89. ECS1 expression highly sensitizes cells to lysosomotropic agents and microtubule-destabilizing drugs.

      Expression of RECS1 causes an acute response of cells to lysosomal and microtubule stress, leading to cell death

    90. cytotoxic agents

      Chemicals that may be toxic to cells at certain doses

    91. TMBIM family, RECS1 expression results in cell death via the mitochondrial pathway of apoptosis.

      RECS1 is pro cell death, although other members of TMBIM family were reported as inhibitors of cell death

    92. gain- and loss-of-function approaches

      Enhance (gain) or reduce/block (loss) the expression of a gene to study its biological function

    93. intracellular

      Inside the cell

    94. Additional sequence analyses revealed the presence of a conserved di-aspartyl sensor and an arginine latch, shown to be critical for the regulation of the calcium channel function of the bacterial (Bacillus subtilis) homolog BsYetJ and human GAAP (11, 12, 25).

      RECS1 has features (di-aspartyl sensor and an arginine latch) found in related proteins in bacteria and humans, which are well investigated.

    95. Golgi apparatus

      One of the cell organelles

    96. BH3-like motif

      A segment of RECS1 that is thought to positively regulate cell death

    97. However, no proapoptotic molecules have been identified in the mammalian TMBIM family.

      Does mammalian TMBIM family of proteins exhibit proapoptotic properties?

    98. ortholog

      A similar gene in different species of organisms

    99. putative

      Thought to be

    100. rheostat

      Regulation by control of the levels of contrasting signals

    101. RECS1 and LFG are regarded as inhibitors of the extrinsic pathway of apoptosis as they block cell death induced by Fas ligand (18, 19), while BI-1, GRINA, and GAAP repress apoptosis induced by intracellular cytotoxic stimuli (10, 17).

      Here, RECS1 was reported to be an inhibitor (negative regulator) of apoptosis.

    102. ion channe

      A passage in the cell membranes through which charged chemical elements (ions) enter or exit the cell or cell compartment

    103. calcium homeostasis

      Regulation of Calcium use in cells.

    104. In addition to BI-1, the TMBIM superfamily includes the following members: responsive to centrifugal force and shear stress 1 (RECS1/TMBIM1), lifeguard (LFG/TMBIM2), glutamate receptor ionotropic N-methyl-D-aspartate receptor 1 (GRINA/TMBIM3), Golgi anti-apoptotic protein (GAAP/TMBIM4), and growth hormone–inducible protein (GHITM/TMBIM5) (8).

      All these proteins negatively regulate programmed cell death

    105. cytotoxic stimuli

      Cyto (from cell) - toxic: Poisonous situation/condition/change in a cell

    106. homologs

      Related proteins across different organisms

    107. transmembrane BAX inhibitor motif containing (TMBIM) superfamily

      A group of proteins that span membranes and oppose the function of BAX (a positive regulator of apoptosis).

    108. pathophysiological


    109. lysosomal membrane permeabilization (LMP)

      Making a membrane easily take up external substances, where it would normally not

    110. caspase cascade

      A group of proteins that function together in programmed cell death

    111. cytochrome c

      Protein associated with the inner membrane of mitochondria and is involved in apoptosis

    112. homo-oligomerization

      Protein complex consisting of identical protein subunits.

    113. conformational activation

      Activation preceded by a change in protein structure

    114. proapoptotic proteins

      Positively regulate programmed cell death. Proteins such as BAK and BAX

    115. to ER stress. RECS1 overexpression resulted in reduced survival when zebrafish were exposed to Tg in the water, while overexpression of the mouse GRINA conferred protection (Fig. 10, H and I) as reported (17). W

      Zebrafish expressing high dose of RECS1 exhibit reduced survival under ER stress

    116. Dysmorphic embryos

      Zebrafish embryos with abnormal morphology/structure/shape.

    117. Next, we determined the importance of RECS1 channel activity to cell death control during zebrafish development. We injected single-cell embryos with RECS1 WT or the D295Q mutant and assessed animal viability 24 hpf. In line with our in vitro results, overexpression of the RECS1 D295Q mutant failed to induce cell death in these embryos (Fig. 10E). Moreover, while the injection of RECS1 WT resulted in a high proportion of morphological-altered embryos, expression of the mutant protein did not have any adverse effect (Fig. 10, F and G).

      The ion channel activity of RECS1 (through D295) is essential in regulating cell death in Zebrafish.

    118. As expected, RECS1 overexpression induced a high number of AO spots, distributed along the head and, to a lesser extent, the body of the embryo (Fig. 10, C and D). Deletion of the C-terminal domain of RECS1 completely abrogated its cytotoxic effects (Fig. 10, C and D). T

      Relative to whole body, head cells were more affected by a high dose of RECS1. Cell death was also dependent on the presence of C-terminal segment of RECS1.

    119. cridine orange (

      An organic dye for cell imaging. Dead cells are stained more by AO.

    120. RECS1 overexpression significantly decreased animal survival, a phenotype that was independent of its C-terminal domain (Fig. 10A). As a control, we overexpressed the TMBIM protein GRINA, which, under the same conditions, did not result in any developmental defects (Fig. 10A).

      High dose of RECS1 is associated with reduced survival in Zebrafish. The effect of RECS1 here seems to not require its C-terminal domain unlike in mammalian cells in which the authors reported that the RECS1 C-terminal is necessary for RECS1-mediated cell death (Supplementary figure 1).

    121. Together, these results suggest that the C-terminal region of RECS1 is essential to induce cell death, but this sequence does not bear functional bona fide BH3 motifs.

      Is there a minimum peptide sequence length within the C-terminal below which the C-terminal is irrelevant in RECS1-regulated cell death?

    122. itochondrial potential (ΔΨm)–sensitive dye JC-1

      JC-1 changes when mitochondria charge changes

    123. o investigate the role of RECS1 on the control of cell death in vivo in a vertebrate model, we overexpressed the full-length human RECS1 in zebrafish by injecting in vitro synthetized mRNA to one-cell stage embryos, followed by the analysis of animal viability and morphology 24 hours post-fertilization (hpf)

      The authors expressed a high dose of human RECS1 in Zebrafish embryo. It is expected that the human RECS1 will have similar function in Zebrafish, as this protein function is conserved across different organisms.

    124. There is a high degree of functional conservation in the mechanisms regulating cell death between vertebrates, with almost all the members of the BCL-2 and the TMBIM families conserved in zebrafish

      The functions of proteins in BCL-2 and the TMBIM family are similar across multiple organisms.

    125. verexpression of dRECS1 strongly sensitizes to lysosomal perturbations and ER stress, resulting in exacerbated wing abnormalities (Fig. 9A).

      More flies with high dose of RECS1 develop severely abnormal wings when exposed to Tm and CQ, compared to flies that have normal RECS1 dose, suggesting that RECS1 leads to abnormal development under stress conditions.

    126. The di-aspartyl sensor is conserved in all six TMBIM proteins, while the arginine latch is only present in the LFG subfamily (LFG1 to LFG4) but not in BI-1, where it has been replaced by His78 (Fig. 7A) (11).

      Two conserved Aspartic acid residues in all proteins in this group. These two residues are involved in sensing lysosomal pH changes.

    127. BsYetJ has 27% identity with GAAP and 36% with RECS1 (fig. S7). In the bacterial BsYetJ, the closed channel conformation is maintained through the interaction of the Arg60 with a conserved di-aspartyl group formed by Asp171-Asp195, which are hydrogen-bonded to one another through their carboxylate groups

      The sequence and structure of the mammalian RECS1 is closely related to the bacteria BsYetJ

    128. intraluminal cathepsins

      Proteins inside the lysosomes that degrade other proteins

    129. annexin V

      a dye for detecting apoptotic cells

    130. BAX has been shown to translocate to lysosomes where it is suggested to trigger LMP and cell death under different pathophysiological conditions, including Parkinson’s disease, oxidative stress, and autophagic cell death (35–37). To assess BAX translocation to the lysosomal membrane and its colocalization with RECS1, we detected active BAX using the 6A7 conformational antibody. Analysis of RECS1 (using the Flag antibody) and the distribution of the lysosomal protein LAMP-2 in MEFs indicated that under basal conditions (no RECS1), BAX remained mainly cytosolic (fig. S4H). However, BAX colocalized with RECS1 in LAMP-2–positive vesicles following RECS1 induction (Fig. 3, E to G). After CQ treatment, BAX was redistributed into large LAMP-2–positive vesicles, an effect that was dependent on RECS1 expression (Fig. 3E). Active BAX was present preferentially in Flag-RECS1–positive lysosomes in cells treated with CQ (Fig. 3, F and G). Together, these results suggest that RECS1 induces cell death through LMP in response to lysosomal stress, correlating with the translocation of BAX to lysosomes

      Overexpression of RECS1 in CQ-stressed cells causes LMP and translocation of BAX to the lysosome, which induces cell death. BAX is a proapoptotic protein.

    131. cytotoxic

      Toxic to cells at certain doses

    132. abrogate


    133. Ablation

      To remove

    134. BAX and BAK

      Two of the proteins that are well known to be activators of cell death (proapoptotic proteins)

    135. SYTO16 staining

      A green fluorescent dye that stains live cells

    136. lysosomotropic agents

      Chemicals that cause stress to lysosomes