A multi-body dynamics approach for the study of critical handling manoeuvres on surfaces with uneven friction

The study of the dynamic behaviour of vehicles using computer simulation has been one of the major areas of research for many years. Based on the application area, the models used for performing these studies vary greatly in their capability, complexity and amount of data required. The multi-body approach is most preferred when it comes to iterative design optimization, whereas relatively simple models are mostly used for studying basic handling characteristics and vehicle stability. However, for studies involving critical handling manoeuvres, it is imperative to include certain amount of detail in the vehicle model, which accounts for the influence of suspension geometry and tyre characteristics on handling behaviour. The aim of the present research is to develop a vehicle model, based on Newton-Euler formulation of equations, incorporating sufficient degrees of freedom and adequate non linear characteristics for the realistic simulation of severe handling manoeuvres. The model is verified against experimental vehicle data and is finally used for the investigation of critical handling manoeuvres on surfaces with uneven friction. During this procedure, the tendency of the vehicle to rollover is assessed, together with other dynamic outputs such as yaw velocity and lateral acceleration

Influence of tyre transience on anti-lock braking

Transient tyre characteristics can have significant influence in vehicle handling, particularly in anti-lock braking system (ABS), which involves wheel speed oscillations as a result of rapid changes in wheel brake pressure. Hitherto, ABS studies have been conducted mostly with straight-line motion. Relatively simple vehicle models have been used which cannot capture the interactions between non-linear handling dynamics and tyre behaviour. This article takes such interactions into account, using a non-linear 14-degrees-of-freedom vehicle model in combination with three different single-point contact tyre models with transient characteristics. They include a stretched-string-based model, a modified stretched-string model, and a contact mass model. The particularly demanding situation of combined cornering/ABS braking is investigated. It is shown that although all tyre models are of similar bandwidth (maximum frequency ≈ 15 Hz), the simple string tyre model fails to cope with the non-linearities involved in combined braking/cornering and predicts greater braking distances than the two more enhanced tyre models

<i>Lrpprc2</i> mutants show UPS mediated Marf degradation.

(A-C’) lrpprc2A mutant clones (non green cells, A, B and C and dashed white line, A’, B’ and C’), wing discs immunostained for Marf::HA (red) after feeding larvae with DMSO(A-A’), chloroquine(B-B’) or MG132(C-C’). (D-D’) lrpprc2A mutant clones (non green cells, D and dashed white line, D’) on overexpression of Prosβ61 under Actin>Gal4, wing discs immunostained for Marf::HA (red). Scale bar represents 20μm. (A”, B”, C” and D”) Quantification for relative fluorescence intensities of Marf::HA in lrpprc2A mutant clones on treatment with, DMSO (A”,n = 24), chloroquine (B”,n = 23), MG132 (C”, n = 10) and on overexpression of Prosβ61 under Actin>Gal4 (D”,n = 15). Graphs represent intensity values normalized to that of control cells. Two tailed unpaired t-test between control and lrpprc2A mutant cells. Significance represented by n.s. non significant, p<0.001***.</p

<i>Drosophila</i> genotypes used in the study.

Cells under mitochondrial stress often co-opt mechanisms to maintain energy homeostasis, mitochondrial quality control and cell survival. A mechanistic understanding of such responses is crucial for further insight into mitochondrial biology and diseases. Through an unbiased genetic screen in Drosophila, we identify that mutations in lrpprc2, a homolog of the human LRPPRC gene that is linked to the French-Canadian Leigh syndrome, result in PINK1-Park activation. While the PINK1-Park pathway is well known to induce mitophagy, we show that PINK1-Park regulates mitochondrial dynamics by inducing the degradation of the mitochondrial fusion protein Mitofusin/Marf in lrpprc2 mutants. In our genetic screen, we also discover that Bendless, a K63-linked E2 conjugase, is a regulator of Marf, as loss of bendless results in increased Marf levels. We show that Bendless is required for PINK1 stability, and subsequently for PINK1-Park mediated Marf degradation under physiological conditions, and in response to mitochondrial stress as seen in lrpprc2. Additionally, we show that loss of bendless in lrpprc2 mutant eyes results in photoreceptor degeneration, indicating a neuroprotective role for Bendless-PINK1-Park mediated Marf degradation. Based on our observations, we propose that certain forms of mitochondrial stress activate Bendless-PINK1-Park to limit mitochondrial fusion, which is a cell-protective response.</div

S4 Fig -

(A-A’) lrpprc2A mutant clones (non green cells, A and dashed white line, A’), wing discs immunostained for Hsp60 (red). (B-D’) lrpprc2A mutant clones (non green cells, B,C, D and dashed white line, B’ C’, D’) on knockdown of crc(B-B’), foxo(C-C’) and dve(D-D’) using En>Gal4, wing discs marked by UAS-RFP (green) and immunostained for Marf::HA (red). (E-E’) Overexpression of ΔOTC using En>Gal4, wing discs marked by UAS-RFP (green) and immunostained for Hsp60 (red). (F-G”) Overexpression of OTC(F-F’) and ΔOTC(G-G’) using En>Gal4, wing discs marked by UAS-RFP (green) and immunostained for Marf::HA (red). Scale bar represents 20μm. (A”-G”) Quantification for relative fluorescence intensities of Hsp60 in lrpprc2A mutant clones (A”, n = 14), Marf::HA in lrpprc2A mutant clones on knockdown of crc(B”, n = 6), foxo(C”,n = 8) and dve(D”,n = 10), Hsp60 on UAS-ΔOTC expression (E”, n = 6) and Marf::HA on UAS-OTC expression (F”, n = 9) and UAS-ΔOTC expression (G”,n = 18). Graphs represent average intensity values normalized to that of control cells. Two-tailed unpaired t-test between control and lrpprc2A mutant cells/ cells overexpressing UAS-OTC or UAS-ΔOTC. Significance represented by n.s.—non significant, p (TIF)</p

Antibodies used in this study.

Cells under mitochondrial stress often co-opt mechanisms to maintain energy homeostasis, mitochondrial quality control and cell survival. A mechanistic understanding of such responses is crucial for further insight into mitochondrial biology and diseases. Through an unbiased genetic screen in Drosophila, we identify that mutations in lrpprc2, a homolog of the human LRPPRC gene that is linked to the French-Canadian Leigh syndrome, result in PINK1-Park activation. While the PINK1-Park pathway is well known to induce mitophagy, we show that PINK1-Park regulates mitochondrial dynamics by inducing the degradation of the mitochondrial fusion protein Mitofusin/Marf in lrpprc2 mutants. In our genetic screen, we also discover that Bendless, a K63-linked E2 conjugase, is a regulator of Marf, as loss of bendless results in increased Marf levels. We show that Bendless is required for PINK1 stability, and subsequently for PINK1-Park mediated Marf degradation under physiological conditions, and in response to mitochondrial stress as seen in lrpprc2. Additionally, we show that loss of bendless in lrpprc2 mutant eyes results in photoreceptor degeneration, indicating a neuroprotective role for Bendless-PINK1-Park mediated Marf degradation. Based on our observations, we propose that certain forms of mitochondrial stress activate Bendless-PINK1-Park to limit mitochondrial fusion, which is a cell-protective response.</div

Ben is required for maintaining mitochondrial morphology in <i>lrpprc2</i> mutants.

(A-D) Wing disc stained for Mitotracker Red (red) and imaged live for control(A), benA(B), lrpprc2A(C) and lrpprc2A benA(D) mutant clones in peripodial cells. White dashed lines mark aggregated mitochondria and yellow dashed lines mark ring shaped mitochondria. Scale bar represents 5μm. (E) Dot plot representing the number of ring shaped mitochondria present in one peripodial cell, the center line represents the mean value (n = 18). (F) Dot plot representing the number of large aggregated mitochondria present in one peripodial cell, the center line represents the mean value (n = 18). Error bars represent S.E.M. A one-way ANOVA-Tukey’s multiple comparison test was used to calculate the significance between the samples in graph (E) and (F). Significance represented by n.s—non significant, p(G-J) Confocal sections of third instar larval muscles immunostained for Complex V (gray) in control(G), benA(H), lrpprc2A(I) and lrpprc2A benA(J) larvae. Representative individual mitochondrial morphology is marked by different colors: filamentous (red), large globular (yellow) and ring (blue). Scale bar represents 5μm.</p

S6 Fig -

(A-A’) Wing discs immunostained for Marf::mCherry (red) on overexpression of HA::Ben using En>Gal4, wing discs marked with UAS-GFP (green). (A”) Quantification for relative fluorescence intensities of Marf::mCherry on overexpression of HA::ben (n = 12). Graphs represent average intensity values normalized to that of control cells. Two-tailed unpaired t-test between control and UAS-HA::Ben overexpressing cells. n.s—non significant. (TIF)</p

S8 Fig -

(A-D) Confocal sections of third instar larval muscles immunostained for endogenous Complex V (gray) in control(A), benA(B), lrpprc2A(C) and lrpprc2A benA(D) larvae. (TIF)</p

S7 Fig -

(A-A’) benA mutant on overexpression of Pink1 using En>Gal4, wing discs marked with UAS-RFP (blue) and immunostained for Marf::HA (red). (B-B’) benA mutant clone (non-green cell, B and dashed white line, B’), pupal gut 2h APF expressing Sq>mito-EYFP (red). (C-C’) Overexpression of Pink1 using En>Gal4, wing discs marked with UAS-RFP (blue) and immunostained for Complex V (red). (D-D’) Overexpression of Park using En>Gal4, wing discs immunostained for HA (green) and Complex V (red). Scale bar represents 20μm. (C” and D”) Quantification for relative fluorescence intensities of Complex V in UAS-Pink1 cells (C”, n = 15) and UAS-HA::Park cells (D”, n = 15). Graphs represent average intensity values normalized to that of control. Two-tailed unpaired t-test between control and cells overexpressing UAS-Pink1/UAS-HA::Park. (E-E”) Overexpression of Pink1 using En>Gal4, wing discs marked with UAS-RFP (blue, E) and immunostained for Marf::HA (red, E-E”) with lrpprc2A mutant clone (non-green cell, E, E’ and E” and dashed white line, E’ and E”). (F) Average Marf::HA intensity values in wildtype, lrpprc2A mutant clones, UAS-Pink1 and lrpprc2A mutant clones in UAS-Pink1 background, normalized to that of control cells (non RFP expressing GFP positive cells). A one-way ANOVA-Bonferroni’s multiple comparison test was used to calculate the significance between the samples in graph F. Error bars represent S.E.M. Significance represented by n.s.- non significant, p (TIF)</p