Periodic Reporting for period 1 - DILEMMA (DecIphering nucLEar Mechanics in diabetes: a Multi-scAle perspective)
Periodo di rendicontazione: 2022-06-01 al 2024-05-31
Sintesi del contesto e degli obiettivi generali del progetto
Defective molecular, cellular and tissue mechanics are emerging hallmarks for several pathologies. Clinical studies associate diabetes mellitus (DM) to stiffening in vascular, muscle and connecting tissues, determining a particular mechanical fingerprint for this disease. DM is a chronic disease characterized by hyperglycemia, leading to accumulation of irreversible damage to cellular components. At molecular level, high glucose and its by-products determine the non-enzymatic addition of sugar residues to proteins and formation of advanced glycation end products (AGEs). AGEs are clinical biomarkers of DM that correlate with heart failure risk, but the mechanism of action bridging alterations at molecular and tissue level remains elusive. An in-depth multiscale characterization of the DM mechanical landscape in vivo and in vitro is crucial to define new mechanical biomarkers that can lead to better diagnostic, prognostic, and therapeutic strategies. This requires the use of approaches that reunite multiple scales relevant for mechanobiology, from proteins to cells, tissues.
AGE-mediated crosslinking of extracellular matrix (ECM) proteins is considered as the main contributor to tissue stiffness in DM. However, circulatory cells from DM patients also have impaired deformability despite a lack of ECM. Thus, changes in ECM stiffness can only partially explain the stiffening of multiple tissue types and complex physiopathological effects in DM. Alternative mechanisms suggest that direct glycation of intracellular proteins by methylglyoxal (MG), a highly reactive dicarbonyl by-product of glycolysis that increases in DM, induces posttranslational modifications (PTM) in proteins through irreversible nonenzymatic reactions with Arg and Lys residues. Resulting AGEs cause protein loss-of-function and cell dysfunction as shown for intermediate filament (IF) protein vimentin and myofilament tropomyosin.The cell nucleus is an unexpected target for AGE modifications in DM, but recent studies show that MG glycation of the transcriptional cofactors6 and histone residues compromises cell function. The effects of AGE glycation and crosslinking on cell nuclei are of high importance, since the nucleus acts as central hub in cellular mechanotransduction, both structurally and biochemically, with important implications in physiology and disease. In particular, lamins are relevant targets of AGE modifications due to their role in nuclear envelope mechano-regulation, but it is still unclear how the nuclear lamina conserves mechanical functions following AGE modifications common in DM. Here, we worked under the hypothesis that besides the ECM and the cytoskeleton, cellular nuclei can also be affected by stiffening in DM pathology, as a consequence of lamin isoforms glycation. Lamin A was studied as glycation and crosslinking target, with groundbreaking implications in DM biology due to the key role of this isoform in nuclear mechanotransduction, regulation of gene expression, transcriptional activity, and chromatin organization. Lamin glycation has multifold implications, as it could be responsible for enhanced vascular dysfunction and atherosclerosis in DM patients, or play a role in enigmatic single gene mutations causing onset of DM.
The ambition of this interdisciplinary project is to decipher nuclear mechanical alterations in DM using a unique multiscale approach. Due to the unexplored connection between protein biochemistry and biophysical tools, very little is known about the mechanical landscape of the cell nucleus in DM, specifically regarding the possible modifications of lamin A proteins. The DILEMMA project proposes a unique approach that bridges the gap between advanced nanoscopy methods, cell biology and biochemical assays to generate fundamental insights of nuclear mechanics in DM at the nanoscale. We mostly focused on force transduction in lamin A proteins at the single-molecule and cellular level in the context of DM-induced glycation modifications.
AGE-mediated crosslinking of extracellular matrix (ECM) proteins is considered as the main contributor to tissue stiffness in DM. However, circulatory cells from DM patients also have impaired deformability despite a lack of ECM. Thus, changes in ECM stiffness can only partially explain the stiffening of multiple tissue types and complex physiopathological effects in DM. Alternative mechanisms suggest that direct glycation of intracellular proteins by methylglyoxal (MG), a highly reactive dicarbonyl by-product of glycolysis that increases in DM, induces posttranslational modifications (PTM) in proteins through irreversible nonenzymatic reactions with Arg and Lys residues. Resulting AGEs cause protein loss-of-function and cell dysfunction as shown for intermediate filament (IF) protein vimentin and myofilament tropomyosin.The cell nucleus is an unexpected target for AGE modifications in DM, but recent studies show that MG glycation of the transcriptional cofactors6 and histone residues compromises cell function. The effects of AGE glycation and crosslinking on cell nuclei are of high importance, since the nucleus acts as central hub in cellular mechanotransduction, both structurally and biochemically, with important implications in physiology and disease. In particular, lamins are relevant targets of AGE modifications due to their role in nuclear envelope mechano-regulation, but it is still unclear how the nuclear lamina conserves mechanical functions following AGE modifications common in DM. Here, we worked under the hypothesis that besides the ECM and the cytoskeleton, cellular nuclei can also be affected by stiffening in DM pathology, as a consequence of lamin isoforms glycation. Lamin A was studied as glycation and crosslinking target, with groundbreaking implications in DM biology due to the key role of this isoform in nuclear mechanotransduction, regulation of gene expression, transcriptional activity, and chromatin organization. Lamin glycation has multifold implications, as it could be responsible for enhanced vascular dysfunction and atherosclerosis in DM patients, or play a role in enigmatic single gene mutations causing onset of DM.
The ambition of this interdisciplinary project is to decipher nuclear mechanical alterations in DM using a unique multiscale approach. Due to the unexplored connection between protein biochemistry and biophysical tools, very little is known about the mechanical landscape of the cell nucleus in DM, specifically regarding the possible modifications of lamin A proteins. The DILEMMA project proposes a unique approach that bridges the gap between advanced nanoscopy methods, cell biology and biochemical assays to generate fundamental insights of nuclear mechanics in DM at the nanoscale. We mostly focused on force transduction in lamin A proteins at the single-molecule and cellular level in the context of DM-induced glycation modifications.
Lavoro eseguito dall’inizio del progetto fino alla fine del periodo coperto dalla relazione e principali risultati finora ottenuti
Overview of the performed activities:
During the 6 months of the project, I carried out experiments in the framework of Objective 1, Mechanical effect of in vitro lamin glycation. At the same time, I was trained by group members in Jorge Alegre’s team in protein biochemistry and molecular biology methods (by technical assistant Diana Velazquez) and diabetes animal models (by technical assistant Natalia Vicente).
My hypothesis in this Objective 1 that in vitro MG glycation induces nuclear stiffening in cells. Lamin A is one of the main contributors to nuclear mechanotranscution since it mediates the tension-induced nuclear stiffening response in cells and isolated nuclei. I focused on mouse embryonic fibroblasts (MEFs) HeLa cells since they have good lamin expression and are part of connective and myocardial tissues affected by stiffness derived complications of diabetes. My strong background in mechanical profiling of living cells by atomic force microscopy (AFM) enabled me to measure isolated nuclei and nuclear regions of single cells in MG glycation conditions. I simulated the high levels of MG accumulated during DM progression in the body by treating cells with MG concentrations relevant for physiological conditions. To decouple mechanical contributions of other cellular components, in a first set of experiments, nuclei were isolated by membrane disruption with nonionic detergents and probed in AFM indentation experiments to extract Young’s modulus, E.
Scientific activities and main results:
1. Mechanical properties of cells incubated in glycation conditions
To quantify potential changes in cell elasticity modulus, HeLa and MEF cells were used, cultured under control conditions, 0.5mM MG, and 1mM MG for 24 hours. Subsequently, AFM experiments were conducted, in which force-distance curves were recorded in the area of individual cells with 256 curves per cell. The results show that HeLa cells generally have a lower elasticity modulus than MEF cells regardless of glycation conditions; under control conditions, HeLa cells exhibit a median of 1.564 kPa, while for MEF cells, the median is 6.755 kPa, a statistically significant difference (****, p-value <0.0001). Regarding MEF cells, the datasets for each condition also did not follow a normal distribution. Thus, the medians for the 0mM, 0.5mM and 1mM MG conditions are: 6.755 kPa, 5.795 kPa, and 8.96 kPa, respectively. It can be observed that when adding 0.5mM MG to the cell culture, the Young's modulus decreases compared to the control, while it increases when cultured with 1mM MG. Applying the non-parametric Kruskal-Wallis test among them, the differences between the medians of each condition (0, 0.5 and 1 mM MG) are not significant. On the other hand, the datasets for each condition of HeLa cells did meet the assumption of normality, with the means for the 0mM, 0.5mM and 1mM MG conditions being: 1.564 kPa, 1.989 kPa, and 1.01 kPa, respectively. A Welch's ANOVA was conducted on this data and Welch's t-tests were performed between pairs of sets (since they did not meet the assumption of homogeneity of variances), revealing that the differences between the 1mM MG data and the data from the other two conditions were significant (**, p-value < 0.01) while there was no significant difference between those other two conditions.
2. Mechanical properties of isolated nuclei incubated in glycation conditions
To assess the elasticity of isolated nuclei incubated in situ under glycation conditions, isolated HeLa nuclei were immobilized on functionalized glass-bottom plates, where they adhere and maintain their integrity. This allows AFM indentation experiments to be carried out. These experiments were performed at least in triplicate on each nucleus to obtain the average Young's modulus of each nucleus. We monitored the Young’s modulus variation over the incubation time with MG at different concentrations (2mM, 50mM and mock solution). In situ incubation with 50m MG for 360 minutes shows a trend towards increased Young's modulus in nuclei incubated with MG. At time 0, all conditions show values between 50-80 Pa. Under control conditions, the final time values remain within the same range. However, when incubated with 2mM MG, the final Young's modulus values are between 80-110 Pa. The increasing trend becomes quite evident when incubated with 50 mM MG, where final values oscillate between 200-400 Pa. As a result, after 360 minutes, an increase in the slope of the force-distance curves is observed, with the increase being greater as the concentration of MG in the incubation increases.
3. AGEs effects on nuclear morphology
To assess the effects associated with AGEs on nuclear morphology, confocal laser microscopy experiments were conducted. HeLa cells were cultured under control conditions, 0.5mM MG, and 1mM MG for 24 hours, similar to the individual cell AFM experiment. Isolated HeLa nuclei were also incubated in situ under control conditions, 2mM MG, and 50mM MG for 6 hours, similar to the isolated nucleus AFM experiment. DAPI was used as a marker for nuclear structures, binding to DNA, along with anti-lamin A/C antibody, which binds to the nuclear envelope protein.
The results show that under glycation conditions, cells with nuclei of more irregular shapes appear compared to control conditions, where nuclei have more rounded forms. Moreover, a phase separation appears in the images of HeLa nuclei labeled with DAPI, attributed to chromatin condensation. Lamin A/C labeling shows discontinuities in nuclei incubated with MG, which could be due to protein aggregation as a result of glycation. We decided not to evaluate nuclear morphology by marking lamin B1 due to nonspecific labeling of the anti-lamin B1 antibody used.
4. Detection of AGE glycation in lamin A/B and B isoforms
To carry out the primary objective of detecting glycation presence in lamin A/C and B1 isoforms of HeLa and MEF cell and nucleus exposed MG, optimal conditions for lamin extraction were first established. Starting with MEF and HeLa cells cultured in a plate, protein extraction was performed from cells under 12 combinatorial conditions of extraction buffer (TE), protease inhibitor (PI), and addition of N-ethylmaleimide (NEM) / dithiothreitol (DTT). For nuclei with the same objective, 4 combinatorial conditions of TE and addition of NEM/DTT were employed, all with PI. In nuclei, conditions without PI and without NEM/DTT were discarded based on previous laboratory results (without them, a smear appears on the gel, likely due to isolated nuclei being more sensitive to proteases as they are not protected within the cell). The resulting 32 samples were run on 12% SDS-PAGE gels.
The gels show the migration of all proteins extracted from the samples. Focusing on the size range of 60-70 kDa (as lamin A/C has a size of 62/69 kDa, and lamin B1 is 67 kDa), we observe that the use of NEM and DTT can alter protein mobility: with NEM, the band appears at the same size as the sample without NEM or DTT, while with DTT, the band appears at a lower size. In any case, both bands are more defined than those from samples without NEM or DTT. The conditions that showed the best protein extraction performance were selected based on the clarity observed in those bands. The 4 chosen conditions were: TE2 with PI, adding NEM or DTT (arrows in Figure 8), and they were used for the Western Blot detection of lamin A/C and B1. With the selected protein extraction conditions mentioned above, for both complete cells and isolated nuclei of HeLa and MEF, duplicate electrophoresis was repeated on 12% SDS-PAGE gels and transferred to two nitrocellulose membranes. Primary anti-lamin A/C and anti-lamin B1 mouse antibodies were used on them, respectively, followed by secondary anti-mouse antibodies conjugated with HRP for chemiluminescence detection. The membrane for lamin A/C detection shows two bands of a similar size around 75 kDa. Regarding the results for lamin B1, they are inconclusive; bands seem to be obtained only around the expected size (67 kDa) in HeLa nucleus samples, but many nonspecific bands are also present (the membrane is overexposed). Therefore, we cannot draw positive results for lamin B1.
For the detection of AGEs and their colocalization with lamin A/C and B1, proteins were extracted with TE2, PI, and 50 mM DTT from cells cultured under control conditions, 1mM MG, 2mM MG, and 1mM MG + 50mM Glc. The band sizes are reliable and indicate the expected molecular weight. Anti-lamin A/C generates two bands between 50 and 75 kDa (sizes 62 and 69 kDa) and the polyclonal anti-AGEs antibody from Bioss (bs-1158R), which reacts with BSA-AGEs (compounds produced by reaction of bovine serum albumin with glycolaldehyde (GA) (Bioss Antibodies, 2023)), generates a band approximately of the same size as before in HeLa cell samples cultured at 2mM MG and 1mM MG + 50mM Glc. The polyclonal anti-AGEs antibody from Sigma (AB9890), which reacts with proteins modified with GA (Sigma-Aldrich, 2023), leaves a film with a dirty, stained background, and several bands along its surface; As for the rest of the specific anti-AGE antibodies used, the aim was to detect hydroimidazolone, GA-pyrimidine, 3-deoxyglucosone-imidazolone (3-DG-imidazolone), N6-carboxymethyl-lysine (CML), and N6-carboxyethyl-lysine (CEL) as specific modifications of glycating agents. Hydroimidazolone is an AGE resulting from the reaction of MG with an arginine residue; for example, hydroimidazolone-1 (MG-H1) is the most frequent AGE in serum, vascular walls, and the lens (N. Ahmed et al., 2003; Heier et al., 2015). On the other hand, myeloperoxidases catalyze the formation of hypochlorous acid (HOCl); this HOCl can react with serine residues to form glycolaldehyde (GA), which in turn reacts with lysines to generate GA-pyrimidine. This GA-pyrimidine is an AGE present in foam cells and extracellular matrix of atherosclerotic plaques in humans (Nagai et al., 2002). 3-DG-imidazolone is a major AGE in 3-DG-modified proteins, present both intra- and extracellularly in atherosclerotic lesions (Jono et al., 2004).
CML is an AGE found at high levels in the blood of patients with diabetes mellitus. It is associated with the development of heart disease (K. A. Ahmed et al., 2007). CEL is another AGE found in tissues and blood of patients with diabetes mellitus, and along with CML, is a major ligand of RAGE (Xue et al., 2011).
All of these, along with the anti-lamin B1 antibody and the polyclonal antibody from Abcam (ab23722) that detects various human AGEs, have tested negative. To verify the effectiveness of the transfer, both gels that had been transferred were stained with Coomassie blue, as well as the membranes themselves with MemCode™. It can be observed that protein residues remain in the gels without transfer, but in the membranes, the majority have been transferred, confirming a successful transfer.
During the 6 months of the project, I carried out experiments in the framework of Objective 1, Mechanical effect of in vitro lamin glycation. At the same time, I was trained by group members in Jorge Alegre’s team in protein biochemistry and molecular biology methods (by technical assistant Diana Velazquez) and diabetes animal models (by technical assistant Natalia Vicente).
My hypothesis in this Objective 1 that in vitro MG glycation induces nuclear stiffening in cells. Lamin A is one of the main contributors to nuclear mechanotranscution since it mediates the tension-induced nuclear stiffening response in cells and isolated nuclei. I focused on mouse embryonic fibroblasts (MEFs) HeLa cells since they have good lamin expression and are part of connective and myocardial tissues affected by stiffness derived complications of diabetes. My strong background in mechanical profiling of living cells by atomic force microscopy (AFM) enabled me to measure isolated nuclei and nuclear regions of single cells in MG glycation conditions. I simulated the high levels of MG accumulated during DM progression in the body by treating cells with MG concentrations relevant for physiological conditions. To decouple mechanical contributions of other cellular components, in a first set of experiments, nuclei were isolated by membrane disruption with nonionic detergents and probed in AFM indentation experiments to extract Young’s modulus, E.
Scientific activities and main results:
1. Mechanical properties of cells incubated in glycation conditions
To quantify potential changes in cell elasticity modulus, HeLa and MEF cells were used, cultured under control conditions, 0.5mM MG, and 1mM MG for 24 hours. Subsequently, AFM experiments were conducted, in which force-distance curves were recorded in the area of individual cells with 256 curves per cell. The results show that HeLa cells generally have a lower elasticity modulus than MEF cells regardless of glycation conditions; under control conditions, HeLa cells exhibit a median of 1.564 kPa, while for MEF cells, the median is 6.755 kPa, a statistically significant difference (****, p-value <0.0001). Regarding MEF cells, the datasets for each condition also did not follow a normal distribution. Thus, the medians for the 0mM, 0.5mM and 1mM MG conditions are: 6.755 kPa, 5.795 kPa, and 8.96 kPa, respectively. It can be observed that when adding 0.5mM MG to the cell culture, the Young's modulus decreases compared to the control, while it increases when cultured with 1mM MG. Applying the non-parametric Kruskal-Wallis test among them, the differences between the medians of each condition (0, 0.5 and 1 mM MG) are not significant. On the other hand, the datasets for each condition of HeLa cells did meet the assumption of normality, with the means for the 0mM, 0.5mM and 1mM MG conditions being: 1.564 kPa, 1.989 kPa, and 1.01 kPa, respectively. A Welch's ANOVA was conducted on this data and Welch's t-tests were performed between pairs of sets (since they did not meet the assumption of homogeneity of variances), revealing that the differences between the 1mM MG data and the data from the other two conditions were significant (**, p-value < 0.01) while there was no significant difference between those other two conditions.
2. Mechanical properties of isolated nuclei incubated in glycation conditions
To assess the elasticity of isolated nuclei incubated in situ under glycation conditions, isolated HeLa nuclei were immobilized on functionalized glass-bottom plates, where they adhere and maintain their integrity. This allows AFM indentation experiments to be carried out. These experiments were performed at least in triplicate on each nucleus to obtain the average Young's modulus of each nucleus. We monitored the Young’s modulus variation over the incubation time with MG at different concentrations (2mM, 50mM and mock solution). In situ incubation with 50m MG for 360 minutes shows a trend towards increased Young's modulus in nuclei incubated with MG. At time 0, all conditions show values between 50-80 Pa. Under control conditions, the final time values remain within the same range. However, when incubated with 2mM MG, the final Young's modulus values are between 80-110 Pa. The increasing trend becomes quite evident when incubated with 50 mM MG, where final values oscillate between 200-400 Pa. As a result, after 360 minutes, an increase in the slope of the force-distance curves is observed, with the increase being greater as the concentration of MG in the incubation increases.
3. AGEs effects on nuclear morphology
To assess the effects associated with AGEs on nuclear morphology, confocal laser microscopy experiments were conducted. HeLa cells were cultured under control conditions, 0.5mM MG, and 1mM MG for 24 hours, similar to the individual cell AFM experiment. Isolated HeLa nuclei were also incubated in situ under control conditions, 2mM MG, and 50mM MG for 6 hours, similar to the isolated nucleus AFM experiment. DAPI was used as a marker for nuclear structures, binding to DNA, along with anti-lamin A/C antibody, which binds to the nuclear envelope protein.
The results show that under glycation conditions, cells with nuclei of more irregular shapes appear compared to control conditions, where nuclei have more rounded forms. Moreover, a phase separation appears in the images of HeLa nuclei labeled with DAPI, attributed to chromatin condensation. Lamin A/C labeling shows discontinuities in nuclei incubated with MG, which could be due to protein aggregation as a result of glycation. We decided not to evaluate nuclear morphology by marking lamin B1 due to nonspecific labeling of the anti-lamin B1 antibody used.
4. Detection of AGE glycation in lamin A/B and B isoforms
To carry out the primary objective of detecting glycation presence in lamin A/C and B1 isoforms of HeLa and MEF cell and nucleus exposed MG, optimal conditions for lamin extraction were first established. Starting with MEF and HeLa cells cultured in a plate, protein extraction was performed from cells under 12 combinatorial conditions of extraction buffer (TE), protease inhibitor (PI), and addition of N-ethylmaleimide (NEM) / dithiothreitol (DTT). For nuclei with the same objective, 4 combinatorial conditions of TE and addition of NEM/DTT were employed, all with PI. In nuclei, conditions without PI and without NEM/DTT were discarded based on previous laboratory results (without them, a smear appears on the gel, likely due to isolated nuclei being more sensitive to proteases as they are not protected within the cell). The resulting 32 samples were run on 12% SDS-PAGE gels.
The gels show the migration of all proteins extracted from the samples. Focusing on the size range of 60-70 kDa (as lamin A/C has a size of 62/69 kDa, and lamin B1 is 67 kDa), we observe that the use of NEM and DTT can alter protein mobility: with NEM, the band appears at the same size as the sample without NEM or DTT, while with DTT, the band appears at a lower size. In any case, both bands are more defined than those from samples without NEM or DTT. The conditions that showed the best protein extraction performance were selected based on the clarity observed in those bands. The 4 chosen conditions were: TE2 with PI, adding NEM or DTT (arrows in Figure 8), and they were used for the Western Blot detection of lamin A/C and B1. With the selected protein extraction conditions mentioned above, for both complete cells and isolated nuclei of HeLa and MEF, duplicate electrophoresis was repeated on 12% SDS-PAGE gels and transferred to two nitrocellulose membranes. Primary anti-lamin A/C and anti-lamin B1 mouse antibodies were used on them, respectively, followed by secondary anti-mouse antibodies conjugated with HRP for chemiluminescence detection. The membrane for lamin A/C detection shows two bands of a similar size around 75 kDa. Regarding the results for lamin B1, they are inconclusive; bands seem to be obtained only around the expected size (67 kDa) in HeLa nucleus samples, but many nonspecific bands are also present (the membrane is overexposed). Therefore, we cannot draw positive results for lamin B1.
For the detection of AGEs and their colocalization with lamin A/C and B1, proteins were extracted with TE2, PI, and 50 mM DTT from cells cultured under control conditions, 1mM MG, 2mM MG, and 1mM MG + 50mM Glc. The band sizes are reliable and indicate the expected molecular weight. Anti-lamin A/C generates two bands between 50 and 75 kDa (sizes 62 and 69 kDa) and the polyclonal anti-AGEs antibody from Bioss (bs-1158R), which reacts with BSA-AGEs (compounds produced by reaction of bovine serum albumin with glycolaldehyde (GA) (Bioss Antibodies, 2023)), generates a band approximately of the same size as before in HeLa cell samples cultured at 2mM MG and 1mM MG + 50mM Glc. The polyclonal anti-AGEs antibody from Sigma (AB9890), which reacts with proteins modified with GA (Sigma-Aldrich, 2023), leaves a film with a dirty, stained background, and several bands along its surface; As for the rest of the specific anti-AGE antibodies used, the aim was to detect hydroimidazolone, GA-pyrimidine, 3-deoxyglucosone-imidazolone (3-DG-imidazolone), N6-carboxymethyl-lysine (CML), and N6-carboxyethyl-lysine (CEL) as specific modifications of glycating agents. Hydroimidazolone is an AGE resulting from the reaction of MG with an arginine residue; for example, hydroimidazolone-1 (MG-H1) is the most frequent AGE in serum, vascular walls, and the lens (N. Ahmed et al., 2003; Heier et al., 2015). On the other hand, myeloperoxidases catalyze the formation of hypochlorous acid (HOCl); this HOCl can react with serine residues to form glycolaldehyde (GA), which in turn reacts with lysines to generate GA-pyrimidine. This GA-pyrimidine is an AGE present in foam cells and extracellular matrix of atherosclerotic plaques in humans (Nagai et al., 2002). 3-DG-imidazolone is a major AGE in 3-DG-modified proteins, present both intra- and extracellularly in atherosclerotic lesions (Jono et al., 2004).
CML is an AGE found at high levels in the blood of patients with diabetes mellitus. It is associated with the development of heart disease (K. A. Ahmed et al., 2007). CEL is another AGE found in tissues and blood of patients with diabetes mellitus, and along with CML, is a major ligand of RAGE (Xue et al., 2011).
All of these, along with the anti-lamin B1 antibody and the polyclonal antibody from Abcam (ab23722) that detects various human AGEs, have tested negative. To verify the effectiveness of the transfer, both gels that had been transferred were stained with Coomassie blue, as well as the membranes themselves with MemCode™. It can be observed that protein residues remain in the gels without transfer, but in the membranes, the majority have been transferred, confirming a successful transfer.
Progressi oltre lo stato dell’arte e potenziale impatto previsto (incluso l’impatto socioeconomico e le implicazioni sociali più ampie del progetto fino ad ora)
The results generated in the 6 months that this project was active focus on lamin glycation in cellular environments, specifically HeLa and MEF cells.
HeLa cells are human epithelial cells derived from adenocarcinoma, an immortalized cell line widely used in mechanobiology studies, especially related to nuclei (Guilluy et al., 2014; Krieg et al., 2018; Wallace et al., 2023). Additionally, an immortalized cell line facilitates experiments due to its rapid growth and preservation of phenotype across passages. On the other hand, MEF cells are fibroblasts isolated from mouse embryos on day 13 of development. As a cellular model, fibroblasts are interesting for studying diabetes mellitus (DM) because they are part of the connective tissue, which undergoes stiffness changes in this disease. Previous studies also provide evidence that cytoskeletal proteins in fibroblasts, such as vimentin, can be targets of glycation agents that alter contractility (Kueper et al., 2008).
The extraction of lamin A/C and B1 was carried out from individual HeLa and MEF cells, as well as isolated nuclei. The protocols utilized showed higher protein yield when using extraction buffer 2 (TE2) with protease inhibitor (+PI) and agents like DTT or NEM. Regarding the detection of lamin A/C by chemiluminescence, the results show two bands of similar size around 75 kDa. This size is not far from the expected range (one band corresponding to lamin A at 62 kDa, and another to lamin C at 69 kDa), but the experiment needs to be repeated with gels having the correct proportion of concentrator gel and separating gel.
For the Western blot detection of AGEs co-localizing with lamin A/C and B1, the focus was on lamin A/C since lamin B1 could not be identified with certainty. A positive result is obtained with the polyclonal Bioss antibody, which has demonstrated for the first time that AGEs modify lamin proteins. Lamin A/C co-localizes with glycolaldehyde (GA) modifications in HeLa cells incubated with 2mM MG and 1mM MG + 50mM Glc. This positive result indicates that these concentrations of glycation compounds represent a threshold beyond which further experiments could help confirm if lamin A/C is a target of glycation reactions. The rest of the anti-AGE antibodies (which detect AGEs like hydroimidazolone, CML, CEL, GA-pyrimidine, 3-DG-imidazolone, among others) yield negative results, so a potential improvement for this experiment is to increase the concentration at which they are used (for example, the hydroimidazolone antibody or the polyclonal Sigma anti-AGE antibody have been used at very low dilutions, 1:1000 and 1:2000, respectively). The negative controls used (anti-mouse and anti-rabbit) worked well, so the results obtained with all antibodies except the polyclonal Sigma (goat) are valid. Finally, the gel-to-membrane transfers in this experiment are considered successful, observing that the majority of proteins have transferred on the MemCode™ stained membranes. Similarly, some protein residues remain in the gels without transfer, so longer transfer times could be used in replication experiments.
In terms of the AFM experiments in individual cells, HeLa cells have a much lower Young's modulus than MEF cells in general; under control conditions, HeLa cells present an average value of 1.6 kPa, and MEF cells, 6.8 kPa. This statistically significant difference may be attributed to the fact that tumor cells are inherently softer than non-cancerous cells, as shown in the results of (Zou et al., 2023) when comparing the Young's modulus of non-cancerous breast tissue cells to that of breast cancer cells. Comparing our results with the literature is challenging since values depend on the applied force or indentation depth, and the used tip; for instance, HeLa cells measured with k= 0.01 N/m conical tips, applying a force of 1 nN, show a Young's modulus under control conditions of 7.1±5.6 kPa (Camacho-Fernández et al., 2021).
Regarding the MEF cells specifically, no conclusions can be drawn about changes in the elasticity modulus as statistically significant results were not obtained. However, HeLa cells show a significant decrease in the Young's modulus when cultured for 24 hours with 1mM MG. This aligns with experiments conducted on breast cancer-derived cells under high glucose conditions, in which the Young's modulus significantly decreased compared to the control group (Zou et al., 2023). This further supports the notion that a high-sugar environment contributes to the deformability of tumor cells, which, on the other hand, might promote metastasis (Swaminathan et al., 2011; Xu et al., 2012). Along the same line, invasive cells undergoing epithelial-mesenchymal transition tend to have softer nuclei (Harada et al., 2014). This makes DM a risk factor for tumor development, as indicated by the higher rate of tumor recurrence and metastasis in breast cancer and DM patients compared to breast cancer patients with normal blood glucose levels (Cejuela et al., 2022).
Regarding the AFM experiment on isolated nuclei, the successful immobilization of isolated nuclei on functionalized glass-bottom plates has been achieved, as the nuclei maintain their structural integrity and adhere to the substrate. As for the results, the increasing trend of the Young's modulus in nuclei when incubated with MG compared to control nuclei aligns with expectations, as non-enzymatic chemical modifications like glycation directly correlate with the concentration of reactants and reaction time. These results also coincide with findings in the literature, where tumor cells cultured with MG exhibit high levels of histone glycation in nuclei; this leads to chromatin disassembly and the appearance of cross-linked products in proteins and DNA (Zheng et al., 2019). This study also conducts single-molecule experiments using optical tweezers on nucleosomes, demonstrating that higher forces are required to stretch nucleosomes treated with MG due to the generated cross-links.
Finally, concerning the confocal laser microscopy experiment on HeLa cells and nuclei, changes in nuclear morphology under glycation conditions are observed. Irregular structures with "peaks" appear, contrasting with the rounded morphology under control conditions, as well as a more condensed chromatin structure and aggregation of lamin A/C under glycation conditions, compared to more homogeneous and regular structures under control conditions. These findings also align with the previously provided information on changes in nuclear structures, such as chromatin disassembly and cross-linking in proteins and DNA, upon induction of nuclear glycation (Zheng et al., 2019).
Based on the experiments conducted and the results obtained in this project, we obtained results that go beyond the state-of-the-art, which show that incubation under glycation conditions with methylglyoxal causes:
• The formation of advanced glycation end products (AGEs) is a process that occurs in lamin A/C of HeLa cell nuclear envelopes.
• Variations in the Young's modulus of individual HeLa cells at different exposure concentrations, while MEF cells do not show significant differences.
• A progressive increase in the Young's modulus of isolated HeLa nuclei.
• Changes in nuclear architecture, including aggregation of lamin A/C and chromatin condensation.
• Alteration of nuclear morphology in both HeLa cells and isolated nuclei.
These observations serve as a basis for further investigating the alterations that nuclear lamin undergoes in environments that induce glycation, due to their potential negative consequences on the development of their structural function (nuclear integrity and mechanical stiffness) and mechanotransduction of signals. Subsequent experiments could extend the detection of AGEs in lamin isoforms by refining the experiments conducted in this study, and could even focus on specific modifications and residues using other techniques such as proteomics. Additionally, changes in nuclear morphology could be more comprehensively studied, and it could be determined if tissues extracted from animal models of DM exhibit similar changes as described in this study.
HeLa cells are human epithelial cells derived from adenocarcinoma, an immortalized cell line widely used in mechanobiology studies, especially related to nuclei (Guilluy et al., 2014; Krieg et al., 2018; Wallace et al., 2023). Additionally, an immortalized cell line facilitates experiments due to its rapid growth and preservation of phenotype across passages. On the other hand, MEF cells are fibroblasts isolated from mouse embryos on day 13 of development. As a cellular model, fibroblasts are interesting for studying diabetes mellitus (DM) because they are part of the connective tissue, which undergoes stiffness changes in this disease. Previous studies also provide evidence that cytoskeletal proteins in fibroblasts, such as vimentin, can be targets of glycation agents that alter contractility (Kueper et al., 2008).
The extraction of lamin A/C and B1 was carried out from individual HeLa and MEF cells, as well as isolated nuclei. The protocols utilized showed higher protein yield when using extraction buffer 2 (TE2) with protease inhibitor (+PI) and agents like DTT or NEM. Regarding the detection of lamin A/C by chemiluminescence, the results show two bands of similar size around 75 kDa. This size is not far from the expected range (one band corresponding to lamin A at 62 kDa, and another to lamin C at 69 kDa), but the experiment needs to be repeated with gels having the correct proportion of concentrator gel and separating gel.
For the Western blot detection of AGEs co-localizing with lamin A/C and B1, the focus was on lamin A/C since lamin B1 could not be identified with certainty. A positive result is obtained with the polyclonal Bioss antibody, which has demonstrated for the first time that AGEs modify lamin proteins. Lamin A/C co-localizes with glycolaldehyde (GA) modifications in HeLa cells incubated with 2mM MG and 1mM MG + 50mM Glc. This positive result indicates that these concentrations of glycation compounds represent a threshold beyond which further experiments could help confirm if lamin A/C is a target of glycation reactions. The rest of the anti-AGE antibodies (which detect AGEs like hydroimidazolone, CML, CEL, GA-pyrimidine, 3-DG-imidazolone, among others) yield negative results, so a potential improvement for this experiment is to increase the concentration at which they are used (for example, the hydroimidazolone antibody or the polyclonal Sigma anti-AGE antibody have been used at very low dilutions, 1:1000 and 1:2000, respectively). The negative controls used (anti-mouse and anti-rabbit) worked well, so the results obtained with all antibodies except the polyclonal Sigma (goat) are valid. Finally, the gel-to-membrane transfers in this experiment are considered successful, observing that the majority of proteins have transferred on the MemCode™ stained membranes. Similarly, some protein residues remain in the gels without transfer, so longer transfer times could be used in replication experiments.
In terms of the AFM experiments in individual cells, HeLa cells have a much lower Young's modulus than MEF cells in general; under control conditions, HeLa cells present an average value of 1.6 kPa, and MEF cells, 6.8 kPa. This statistically significant difference may be attributed to the fact that tumor cells are inherently softer than non-cancerous cells, as shown in the results of (Zou et al., 2023) when comparing the Young's modulus of non-cancerous breast tissue cells to that of breast cancer cells. Comparing our results with the literature is challenging since values depend on the applied force or indentation depth, and the used tip; for instance, HeLa cells measured with k= 0.01 N/m conical tips, applying a force of 1 nN, show a Young's modulus under control conditions of 7.1±5.6 kPa (Camacho-Fernández et al., 2021).
Regarding the MEF cells specifically, no conclusions can be drawn about changes in the elasticity modulus as statistically significant results were not obtained. However, HeLa cells show a significant decrease in the Young's modulus when cultured for 24 hours with 1mM MG. This aligns with experiments conducted on breast cancer-derived cells under high glucose conditions, in which the Young's modulus significantly decreased compared to the control group (Zou et al., 2023). This further supports the notion that a high-sugar environment contributes to the deformability of tumor cells, which, on the other hand, might promote metastasis (Swaminathan et al., 2011; Xu et al., 2012). Along the same line, invasive cells undergoing epithelial-mesenchymal transition tend to have softer nuclei (Harada et al., 2014). This makes DM a risk factor for tumor development, as indicated by the higher rate of tumor recurrence and metastasis in breast cancer and DM patients compared to breast cancer patients with normal blood glucose levels (Cejuela et al., 2022).
Regarding the AFM experiment on isolated nuclei, the successful immobilization of isolated nuclei on functionalized glass-bottom plates has been achieved, as the nuclei maintain their structural integrity and adhere to the substrate. As for the results, the increasing trend of the Young's modulus in nuclei when incubated with MG compared to control nuclei aligns with expectations, as non-enzymatic chemical modifications like glycation directly correlate with the concentration of reactants and reaction time. These results also coincide with findings in the literature, where tumor cells cultured with MG exhibit high levels of histone glycation in nuclei; this leads to chromatin disassembly and the appearance of cross-linked products in proteins and DNA (Zheng et al., 2019). This study also conducts single-molecule experiments using optical tweezers on nucleosomes, demonstrating that higher forces are required to stretch nucleosomes treated with MG due to the generated cross-links.
Finally, concerning the confocal laser microscopy experiment on HeLa cells and nuclei, changes in nuclear morphology under glycation conditions are observed. Irregular structures with "peaks" appear, contrasting with the rounded morphology under control conditions, as well as a more condensed chromatin structure and aggregation of lamin A/C under glycation conditions, compared to more homogeneous and regular structures under control conditions. These findings also align with the previously provided information on changes in nuclear structures, such as chromatin disassembly and cross-linking in proteins and DNA, upon induction of nuclear glycation (Zheng et al., 2019).
Based on the experiments conducted and the results obtained in this project, we obtained results that go beyond the state-of-the-art, which show that incubation under glycation conditions with methylglyoxal causes:
• The formation of advanced glycation end products (AGEs) is a process that occurs in lamin A/C of HeLa cell nuclear envelopes.
• Variations in the Young's modulus of individual HeLa cells at different exposure concentrations, while MEF cells do not show significant differences.
• A progressive increase in the Young's modulus of isolated HeLa nuclei.
• Changes in nuclear architecture, including aggregation of lamin A/C and chromatin condensation.
• Alteration of nuclear morphology in both HeLa cells and isolated nuclei.
These observations serve as a basis for further investigating the alterations that nuclear lamin undergoes in environments that induce glycation, due to their potential negative consequences on the development of their structural function (nuclear integrity and mechanical stiffness) and mechanotransduction of signals. Subsequent experiments could extend the detection of AGEs in lamin isoforms by refining the experiments conducted in this study, and could even focus on specific modifications and residues using other techniques such as proteomics. Additionally, changes in nuclear morphology could be more comprehensively studied, and it could be determined if tissues extracted from animal models of DM exhibit similar changes as described in this study.