bims-bicyki 2022-06-12 papers

bims-bicyki

Biomed News

on Bicaudal-C1 and interactors in cystic kidney disease

Issue of 2022‒06‒12
seventeen papers selected by
Céline Gagnieux
École Polytechnique Fédérale de Lausanne (EPFL)

Can ketogenic dietary interventions slow disease progression in ADPKD: what we know and what we don't.
More dissimilarities than affinities between DNAJB11-PKD and ADPKD.
Comparative Efficacy of Pharmacological Treatments for Adults With Autosomal Dominant Polycystic Kidney Disease: A Systematic Review and Network Meta-Analysis of Randomized Controlled Trials.
Simultaneous nephrectomy during kidney transplantation for polycystic kidney disease does not detrimentally impact comorbidity and graft survival.
Ketogenic dietary interventions in autosomal dominant polycystic kidney disease-a retrospective case series study: first insights into feasibility, safety and effects.
Angiomotin isoform 2 promotes binding of PALS1 to KIF13B at primary cilia and regulates ciliary length and signaling.
Ciliary membrane, localised lipid modification and cilia function.
Primary cilia in satellite cells are the mechanical sensors for muscle hypertrophy.
Structural diversity in a stereotypic organelle - Sensory cilia of Caenorhabditis elegans.
Reducing Proteoglycan Synthesis and NOX Activity by ROCK Inhibitors: Therapeutic Targets in Atherosclerosis.
Unilateral Cystic Kidney Disease in a Young Female.
Inhibition of Ciliogenesis Enhances the Cellular Sensitivity to Temozolomide and Ionizing Radiation in Human Glioblastoma Cells.
Preliminary results on a proposed histopathological assessment of predictive factors for basal cell carcinoma recurrence after primary free margin excision.
Inhibition of Rho-associated protein kinase activity enhances oxidative phosphorylation to support corneal endothelial cell migration.
Precise control of microtubule disassembly in living cells.
Polycystin-2 (PC2) is a key determinant of in-vitro myogenesis.
Quantifying cilia beat frequency using high-speed video microscopy: Assessing frame rate requirements when imaging different ciliated tissues.

Clin Kidney J. 2022 Jun;15(6): 1034-1036

Can ketogenic dietary interventions slow disease progression in ADPKD: what we know and what we don't.

Albert C M Ong, Roser Torra.

  Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease leading to kidney failure. To date, there is no cure for the disease although there is one approved disease-modifying therapy: tolvaptan. In this context, a common question that ADPKD patients ask in clinical practice is whether there is anything they can do to slow their disease by modifying their diet or lifestyle. Recent evidence from experimental PKD models has shown the potential benefits of caloric restriction, high water intake and especially ketogenic diets in preserving kidney function. Whether these benefits are translatable to humans remains unknown. In this issue of CKJ, Strubl et al. report results of a self-enrolled survey of autosomal dominant polycystic kidney disease (ADPKD) patients who have self-administered a ketogenic diet [1]. These results provide interesting insights into the tolerability, potential benefits and harms of such an intervention that could inform a future clinical trial.

Keywords:  ADPKD; GFR; disease progression; fasting; ketogenic diets

DOI:  https://doi.org/10.1093/ckj/sfac103
Clin Kidney J. 2022 Jun;15(6): 1179-1187

More dissimilarities than affinities between DNAJB11-PKD and ADPKD.

Isabella Pisani, Marco Allinovi, Viviana Palazzo, Paola Zanelli, Micaela Gentile, Maria Teresa Farina, Sara Giuliotti, Paolo Cravedi, Marco Delsante, Umberto Maggiore, Enrico Fiaccadori, Lucio Manenti.

  Background: Polycystic kidney diseases (PKD) are an important cause of chronic kidney disease (CKD). Autosomal dominant polycystic kidney disease (ADPKD) due to PKD1 or PKD2 mutations is the most common form, but other genes can be responsible for ADPKD and its phenocopies. Among them, a form of atypical ADPKD caused by DNAJB11 mutations (DNAJB11-PKD) has been recently described.Methods: We retrospectively recruited a cohort of 27 patients from six different families sharing common ancestries and harboring the same DNAJB11 mutation (c.100C>T, p.Arg34*) and we compared it with a cohort of 42 typical ADPKD patients.
Results: DNAJB11-PKD patients show small/normal-sized kidneys, with significantly smaller cysts and a slower progression to end-stage kidney disease (ESKD) than ADPKD patients. In the DNAJB11-PKD cohort, the cystic phenotype could not be detected by ultrasound in about half of the patients, but all cases with available computed tomography/magnetic resonance scans displayed cysts. Clinically, DNAJB11-PKD patients displayed proteinuria (mostly albuminuria). Compared with ADPKD, DNAJB11-PKD patients were older and had a higher prevalence of type 2 diabetes mellitus (19% versus 0%; P = 0.007) and nephrolithiasis (62% versus 29%; P = 0.01), whereas the prevalence of cardiac valvular defects was lower (4% versus 51%; P < 0.001).
Conclusions: Overall, clinical features of DNAJB11-PKD were more subtle compared with those of ADPKD. DNAJB11-PKD shows a unique renal and extrarenal phenotype, clinical presentation and natural history. Therefore our data support that this genetic disease is classified separately from ADPKD.

Keywords:  ADPKD; DNAJB11; chronic kidney disease; cystic kidney disease; genetics

DOI:  https://doi.org/10.1093/ckj/sfac032
Front Pharmacol. 2022 ;13 885457

Comparative Efficacy of Pharmacological Treatments for Adults With Autosomal Dominant Polycystic Kidney Disease: A Systematic Review and Network Meta-Analysis of Randomized Controlled Trials.

Shunichiro Tsukamoto, Shingo Urate, Takayuki Yamada, Kengo Azushima, Takahiro Yamaji, Sho Kinguchi, Kazushi Uneda, Tomohiko Kanaoka, Hiromichi Wakui, Kouichi Tamura.

  Background: Tolvaptan is the gold standard treatment for autosomal dominant polycystic kidney disease (ADPKD), while several other drugs have the potential to inhibit the progression of ADPKD. However, individual clinical trials may not show sufficient differences in clinical efficacy due to small sample sizes. Furthermore, the differences in therapeutic efficacy among drugs are unclear. Herein, we investigated the effect of the ADPKD treatments. Methods: We systematically searched PubMed, Medline, EMBASE, and the Cochrane Library through January 2022 to identify randomized controlled trials in ADPKD patients that compared the effects of treatments with placebo or conventional therapy. A network meta-analysis was performed to compare the treatments indirectly. The primary outcomes were changes in kidney function and the rate of total kidney volume (TKV) growth. Results: Sixteen studies were selected with a total of 4,391 patients. Tolvaptan significantly preserved kidney function and inhibited TKV growth compared to the placebo {standardized mean difference (SMD) [95% confidence interval (CI)]: 0.24 (0.16; 0.31) and MD: -2.70 (-3.10; -2.30), respectively}. Tyrosine kinase inhibitors and mammalian target of rapamycin (mTOR) inhibitors inhibited TKV growth compared to the placebo; somatostatin analogs significantly inhibited TKV growth compared to the placebo and tolvaptan [MD: -5.69 (-7.34; -4.03) and MD: -2.99 (-4.69; -1.29), respectively]. Metformin tended to preserve renal function, although it was not significant [SMD: 0.28 (-0.05; 0.61), p = 0.09]. Conclusion: The therapeutic effect of tolvaptan was reasonable as the gold standard for ADPKD treatment, while somatostatin analogs also showed notable efficacy in inhibiting TKV growth. Systematic Review Registration: https://www.crd.york.ac.uk/prospero/, identifier CRD42022300814.

Keywords:  autosomal dominant polycystic kidney disease; kidney function; network meta-analysis; tolvaptan; total kidney volume

DOI:  https://doi.org/10.3389/fphar.2022.885457
World J Transplant. 2022 May 18. 12(5): 100-111

Simultaneous nephrectomy during kidney transplantation for polycystic kidney disease does not detrimentally impact comorbidity and graft survival.

Tom Darius, Sébastien Bertoni, Martine De Meyer, Antoine Buemi, Arnaud Devresse, Nada Kanaan, Eric Goffin, Michel Mourad.

  BACKGROUND: The lack of space, as an indication for a native unilateral nephrectomy for positioning a future kidney graft in the absence of other autosomal dominant polycystic kidney disease-related symptoms, remains controversial.AIM: To evaluate the surgical comorbidity and the impact on graft survival of an associated ipsilateral native nephrectomy during isolated kidney transplantation in patients with autosomal dominant polycystic kidney disease.
METHODS: One hundred and fifty-four kidney transplantations performed between January 2007 and January 2019 of which 77 without (kidney transplant alone (KTA) group) and 77 with associated ipsilateral nephrectomy (KTIN group), were retrospectively reviewed. Demographics and surgical variables were analyzed and their respective impact on surgical comorbidity and graft survival.
RESULTS: Creation of space for future graft positioning was the main reason (n = 74, 96.1%) for associated ipsilateral nephrectomy. No significant difference in surgical comorbidity (lymphocele, wound infection, incisional hernia, wound hematoma, urinary infection, need for blood transfusion, hospitalization stay, Dindo Clavien classification and readmission rate) was observed between the two study groups. The incidence of primary nonfunction and delayed graft function was comparable in both groups [0% and 2.6% (P = 0.497) and 9.1% and 16.9% (P = 0.230), respectively, in the KTA and KTIN group]. The 1- and 5-year graft survival were 94.8% and 90.3%, and 100% and 93.8%, respectively, in the KTA and KTIN group (P = 0.774). The 1- and 5-year patient survival were 96.1% and 92.9%, and 100% and 100%, respectively, in the KTA and KTIN group (P = 0.168).
CONCLUSION: Simultaneous ipsilateral native nephrectomy to create space for graft positioning during kidney transplantation in patients with autosomal dominant polycystic kidney disease does not negatively impact surgical comorbidity and short- and long-term graft survival.

Keywords:  Autosomal dominant polycystic kidney disease; Complications; Graft survival; Kidney transplantation; Surgical comorbidity; Unilateral nephrectomy

DOI:  https://doi.org/10.5500/wjt.v12.i5.100
Clin Kidney J. 2022 Jun;15(6): 1079-1092

Ketogenic dietary interventions in autosomal dominant polycystic kidney disease-a retrospective case series study: first insights into feasibility, safety and effects.

Sebastian Strubl, Simon Oehm, Jacob A Torres, Franziska Grundmann, Jazmine Haratani, Morgan Decker, Sabrina Vuong, Amrit Kaur Bhandal, Nils Methot, Rhianna Haynie-Cion, Franziska Meyer, Florian Siedek, Uwe Korst, Roman-Ulrich Müller, Thomas Weimbs.

  Background: Our laboratory published the first evidence that nutritional ketosis, induced by a ketogenic diet (KD) or time-restricted diet (TRD), ameliorates disease progression in polycystic kidney disease (PKD) animal models. We reasoned that, due to their frequent use for numerous health benefits, some autosomal dominant PKD (ADPKD) patients may already have had experience with ketogenic dietary interventions (KDIs). This retrospective case series study is designed to collect the first real-life observations of ADPKD patients about safety, feasibility and possible benefits of KDIs in ADPKD as part of a translational project pipeline.Methods: Patients with ADPKD who had already used KDIs were recruited to retrospectively collect observational and medical data about beneficial or adverse effects and the feasibility and safety of KDIs in questionnaire-based interviews.
Results: A total of 131 ADPKD patients took part in this study. About 74 executed a KD and 52 a TRD for 6 months on average. A total of 86% of participants reported that KDIs had improved their overall health, 67% described improvements in ADPKD-associated health issues, 90% observed significant weight loss, 64% of participants with hypertension reported improvements in blood pressure, 66% noticed adverse effects that are frequently observed with KDIs, 22 participants reported safety concerns like hyperlipidemia, 45 participants reported slight improvements in estimated glomerular filtration rate and 92% experienced KDIs as feasible while 53% reported breaks during their diet.
Conclusions: Our preliminary data indicate that KDIs may be safe, feasible and potentially beneficial for ADPKD patients, highlighting that prospective clinical trials are warranted to confirm these results in a controlled setting and elucidate the impact of KDIs specifically on kidney function and cyst progression.

Keywords:  autosomal dominant polycystic kidney disease; caloric restriction; intermittent fasting; ketogenic diet

DOI:  https://doi.org/10.1093/ckj/sfab162
J Cell Sci. 2022 Jun 08. pii: jcs.259471. [Epub ahead of print]

Angiomotin isoform 2 promotes binding of PALS1 to KIF13B at primary cilia and regulates ciliary length and signaling.

Stine Kjær Morthorst, Camilla Nielsen, Pietro Farinelli, Zeinab Anvarian, Christina Birgitte R Rasmussen, Andrea Serra-Marques, Ilya Grigoriev, Maarten Altelaar, Nicoline Fürstenberg, Alexander Ludwig, Anna Akhmanova, Søren Tvorup Christensen, Lotte Bang Pedersen.

  The kinesin-3 motor KIF13B functions in endocytosis, vesicle transport, and regulation of ciliary length and signaling. Direct binding of the membrane-associated guanylate kinase (MAGUK) DLG1 to KIF13B's MAGUK-binding stalk (MBS) domain relieves motor autoinhibition and promotes microtubule plus end-directed cargo transport. Here we characterize Angiomotin isoform 2 (Ap80) as a novel KIF13B interactor that promotes binding of another MAGUK, the polarity protein and Crumbs complex component PALS1, to KIF13B. Live-cell imaging analysis indicated that Ap80 is concentrated at and recruits PALS1 to the base of primary cilia, but is not itself a cargo of KIF13B. Consistent with a ciliary function for Ap80, its depletion led to elongated primary cilia and reduced agonist-induced ciliary accumulation of SMO, a key component of the Hedgehog signaling pathway, while Ap80 overexpression caused ciliary shortening. Our results suggest that Ap80 activates KIF13B cargo binding at the base of primary cilia to regulate ciliary length, composition and signaling.

Keywords:  AMOT; Angiomotin p80; Hedgehog signaling; KIF13B; PALS1; Primary cilia

DOI:  https://doi.org/10.1242/jcs.259471
J Cell Physiol. 2022 Jun 06.

Ciliary membrane, localised lipid modification and cilia function.

Priya Dutta, Krishanu Ray.

  Cilium, a tiny microtubule-based cellular appendage critical for cell signalling and physiology, displays a large variety of receptors. The composition and turnover of ciliary lipids and receptors determine cell behaviour. Due to the exclusion of ribosomal machinery and limited membrane area, a cilium needs adaptive logistics to actively reconstitute the lipid and receptor compositions during development and differentiation. How is this dynamicity generated? Here, we examine whether, along with the Intraflagellar-Transport, targeted changes in sector-wise lipid composition could control the receptor localisation and functions in the cilia. We discuss how an interplay between ciliary lipid composition, localised lipid modification, and receptor function could contribute to cilia growth and signalling. We argue that lipid modification at the cell-cilium interface could generate an added thrust for a selective exchange of membrane lipids and the transmembrane and membrane-associated proteins.

Keywords:  Cilia; Ciliopathy; Lipid; Lipid Modification; Signaling

DOI:  https://doi.org/10.1002/jcp.30787
Proc Natl Acad Sci U S A. 2022 Jun 14. 119(24): e2103615119

Primary cilia in satellite cells are the mechanical sensors for muscle hypertrophy.

Weijun Li, Zhenhong Zhu, Kai He, Xiaoyu Ma, Robert J Pignolo, Gary C Sieck, Jinghua Hu, Haitao Wang.

  Skeletal muscle atrophy is commonly associated with aging, immobilization, muscle unloading, and congenital myopathies. Generation of mature muscle cells from skeletal muscle satellite cells (SCs) is pivotal in repairing muscle tissue. Exercise therapy promotes muscle hypertrophy and strength. Primary cilium is implicated as the mechanical sensor in some mammalian cells, but its role in skeletal muscle cells remains vague. To determine mechanical sensors for exercise-induced muscle hypertrophy, we established three SC-specific cilium dysfunctional mouse models-Myogenic factor 5 (Myf5)-Arf-like Protein 3 (Arl3)-/-, Paired box protein Pax-7 (Pax7)-Intraflagellar transport protein 88 homolog (Ift88)-/-, and Pax7-Arl3-/--by specifically deleting a ciliary protein ARL3 in MYF5-expressing SCs, or IFT88 in PAX7-expressing SCs, or ARL3 in PAX7-expressing SCs, respectively. We show that the Myf5-Arl3-/- mice develop grossly the same as WT mice. Intriguingly, mechanical stimulation-induced muscle hypertrophy or myoblast differentiation is abrogated in Myf5-Arl3-/- and Pax7-Arl3-/- mice or primary isolated Myf5-Arl3-/- and Pax7-Ift88-/- myoblasts, likely due to defective cilia-mediated Hedgehog (Hh) signaling. Collectively, we demonstrate SC cilia serve as mechanical sensors and promote exercise-induced muscle hypertrophy via Hh signaling pathway.

Keywords:  exercise; mechanical stimulation; muscle hypertrophy; primary cilia

DOI:  https://doi.org/10.1073/pnas.2103615119
J Cell Physiol. 2022 Jun 10.

Structural diversity in a stereotypic organelle - Sensory cilia of Caenorhabditis elegans.

Ashish K Maurya.

  Sensory cilia, an ancient organelle, displays a high degree of conservation in its structure and functioning. Sensory cilia also fulfill a wide range of sensory functions, from sensing environmental signals (light, sound, chemicals, and mechanical forces) to interpreting intercellular developmental signals. One way they appear to fulfill these diverse and specialized roles is by adopting a variety of shapes and sizes. We are only beginning to document and appreciate this complexity. Here in this review, using the varied and specialized cilia found on Caenorhabditis elegans sensory neurons, I highlight some of the most obvious examples of this structural diversity and the underlying mechanisms if known. Such structural diversity appears to arise from the modulation of deeply conserved molecular pathways and also from cell- and species-specific mechanisms. Studying these ciliary specializations will thus provide for a comprehensive understanding of ciliary biology and might uncover understudied aspects of ciliary disease biology.

Keywords:  Caenorhabditis elegans; cilia; cilia morphology; ciliary diversity

DOI:  https://doi.org/10.1002/jcp.30732
Endocr Metab Immune Disord Drug Targets. 2022 Jun 06.

Reducing Proteoglycan Synthesis and NOX Activity by ROCK Inhibitors: Therapeutic Targets in Atherosclerosis.

Hossein Babaahmadi-Rezaei, Maryam Rezaei, Hossein Ghaderi-Zefrehi, Masoumeh Azizi, Hasti Beheshti-Nasab, Jawahar L Mehta.

  Atherosclerosis is a chronic inflammatory disease of the arteries characterized by accumulation of inflammatory cells in the arterial wall. Hypertension, dyslipidemia, and hyperglycemia are major risk factors of atherosclerosis. Rho-associated protein kinase (ROCK), a serine/threonine kinase, is a downstream effector of the small GTPase RhoA. ROCK is involved in different stages of atherosclerosis. Accumulating evidence has demonstrated that ROCK signaling plays vital roles in various cellular functions, such as contraction, migration, and proliferation of smooth muscle cells. Dysregulation of the ROCK pathway is associated with atherosclerosis and hypertension. Experimental studies have shown that ROCK inhibitors may have favorable effects in ameliorating atherosclerosis. ROCK signaling has a role in proteoglycan synthesis through transactivation of the TGF-β receptor Type I (TβRI) mediated by G-protein-coupled receptor (GPCR) agonists (endothelin-1, angiotensin II and …), and ROCK inhibitors could decrease proteoglycan synthesis and atherosclerotic plaque formation. Based on the hypothesis that targeting ROCK pathway may be effective in ameliorating atherosclerosis, we suggest that ROCK inhibitors may have a potential therapeutic role in inhibition or slowing atherogenesis. However, for this hypothesis more research is needed.

Keywords:  Atherosclerosis; Endothelin-1; NADPH Oxidase; Proteoglycan; ROCK; Transactivation

DOI:  https://doi.org/10.2174/1871530322666220606090801
Cureus. 2022 May;14(5): e24719

Unilateral Cystic Kidney Disease in a Young Female.

Nicholas W Salupo, Megan Grant, Shashikant Patel.

  Unilateral renal cystic disease has been mostly reported in older male patients; however, this case is novel as the youngest reported case in the literature and in a female patient. We present a 22-year-old female with no past medical history and no family history of renal disease that was incidentally found to have unilateral renal cystic disease on computed tomography imaging. The patient's renal function was not impaired and the cystic kidney was found to be functioning appropriately on an intravenous pyelogram. The unilateral cystic disease is benign but must be differentiated from autosomal dominant polycystic disease to prevent morbidity and mortality.

Keywords:  autosomal-dominant polycystic kidney disease; general internal medicine; general nephrology; genetic renal diseases; unilateral renal cystic disease

DOI:  https://doi.org/10.7759/cureus.24719
Biomed Environ Sci. 2022 May 20. 35(5): 419-436

Inhibition of Ciliogenesis Enhances the Cellular Sensitivity to Temozolomide and Ionizing Radiation in Human Glioblastoma Cells.

Li Wei, Wei Ma, Hui Cai, Shao Peng Peng, Huan Bing Tian, Ju Fang Wang, Lan Gao, Jin Peng He.

  Objective: To investigate the function of primary cilia in regulating the cellular response to temozolomide (TMZ) and ionizing radiation (IR) in glioblastoma (GBM).Methods: GBM cells were treated with TMZ or X-ray/carbon ion. The primary cilia were examined by immunostaining with Arl13b and γ-tubulin, and the cellular resistance ability was measured by cell viability assay or survival fraction assay. Combining with cilia ablation by IFT88 depletion or chloral hydrate and induction by lithium chloride, the autophagy was measured by acridine orange staining assay. The DNA damage repair ability was estimated by the kinetic curve of γH2AX foci, and the DNA-dependent protein kinase (DNA-PK) activation was detected by immunostaining assay.
Results: Primary cilia were frequently preserved in GBM, and the induction of ciliogenesis decreased cell proliferation. TMZ and IR promoted ciliogenesis in dose- and time-dependent manners, and the suppression of ciliogenesis significantly enhanced the cellular sensitivity to TMZ and IR. The inhibition of ciliogenesis elevated the lethal effects of TMZ and IR via the impairment of autophagy and DNA damage repair. The interference of ciliogenesis reduced DNA-PK activation, and the knockdown of DNA-PK led to cilium formation and elongation.
Conclusion: Primary cilia play a vital role in regulating the cellular sensitivity to TMZ and IR in GBM cells through mediating autophagy and DNA damage repair.

Keywords:  Autophagy; Cellular sensitivity; DNA damage response; DNA-PK; Glioblastoma; Ionizing radiation; Primary cilia; Temozolomide

DOI:  https://doi.org/10.3967/bes2022.058
Skin Health Dis. 2022 Jun;2(2): e88

Preliminary results on a proposed histopathological assessment of predictive factors for basal cell carcinoma recurrence after primary free margin excision.

A Jacquet, V Dormoy, M Lorenzato, A Durlach.

Background: Basal cell carcinoma (BCC) incidence is steadily increasing but therapeutic solutions remain limited and present a public health challenge.Aims: To identify predictive factors of BCC recurrence after primary free margin excision, with automated methods, by evaluating cell proliferation, the Hedgehog pathway activation and primary cilia.
Materials and Methods: This case-control study included 32 patients (16 with recurrence occurring at least 6 months after complete resection, and 16 without recurrence) who underwent surgery for BCC. Formalin-fixed paraffin-embedded cutaneous resections were processed for immunohistochemistry or immunostaining with the following primary antibodies: mouse anti-MCM6, rabbit anti-ARL13B and rabbit anti-GLI1.
Results: BCC recurrence after free margin excision was significantly linked to a higher proliferative index (p < 0.001) and a lower cilia count (p = 0.041) in the primary lesion. No significant differences were observed regarding cilia length (p = 0.39) or GLI1-positive nuclei.
Discussion: The complex interplay between essential signaling pathways, cell proliferation and cilia requires further experimental investigations in the context of BCC recurrence.
Conclusion: A higher proliferative index evaluated with MCM6 antibody could be a useful prognosis marker of BCC risk of recurrence. The lower cilia count in the primary lesion unveiled novel perspectives to understand BCC recurrence molecular mechanisms.

DOI: https://doi.org/10.1002/ski2.88
FASEB J. 2022 Jul;36(7): e22397

Inhibition of Rho-associated protein kinase activity enhances oxidative phosphorylation to support corneal endothelial cell migration.

Wei-Ting Ho, Jung-Shen Chang, Tsan-Chi Chen, Jia-Kang Wang, Shu-Wen Chang, Muh-Hwa Yang, Tzuu-Shuh Jou, I-Jong Wang.

  Corneal endothelial cell (CEC) dysfunction causes corneal edema and severe visual impairment that require transplantation to restore vision. To address the unmet need of organ shortage, descemetorhexis without endothelial keratoplasty has been specifically employed to treat early stage Fuchs endothelial corneal dystrophy, which is pathophysiologically related to oxidative stress and exhibits centrally located corneal guttae. After stripping off central Descemet's membrane, rho-associated protein kinase (ROCK) inhibitor has been found to facilitate CEC migration, an energy-demanding task, thereby achieving wound closure. However, the correlation between ROCK inhibition and the change in bioenergetic status of CECs remained to be elucidated. Through transcriptomic profiling, we found that the inhibition of ROCK activity by the selective inhibitor, ripasudil or Y27632, promoted enrichment of oxidative phosphorylation (OXPHOS) gene set in bovine CECs (BCECs). Functional analysis revealed that ripasudil, a clinically approved anti-glaucoma agent, enhanced mitochondrial respiration, increased spare respiratory capacity, and induced overexpression of electron transport chain components through upregulation of AMP-activated protein kinase (AMPK) pathway. Accelerated BCEC migration and in vitro wound healing by ripasudil were diminished by OXPHOS and AMPK inhibition, but not by glycolysis inhibition. Correspondingly, lamellipodial protrusion and actin assembly that were augmented by ripasudil became reduced with additional OXPHOS or AMPK inhibition. These results indicate that ROCK inhibition induces metabolic reprogramming toward OXPHOS to support migration of CECs.

Keywords:  AMP-activated protein kinase; cell migration; corneal endothelial cell; mitochondria; oxidative phosphorylation; rho-associated protein kinase

DOI:  https://doi.org/10.1096/fj.202101442RR
EMBO J. 2022 Jun 10. e110472

Precise control of microtubule disassembly in living cells.

Grace Y Liu, Shiau-Chi Chen, Gang-Hui Lee, Kritika Shaiv, Pin-Yu Chen, Hsuan Cheng, Shi-Rong Hong, Wen-Ting Yang, Shih-Han Huang, Ya-Chu Chang, Hsien-Chu Wang, Ching-Lin Kao, Pin-Chiao Sun, Ming-Hong Chao, Yian-Ying Lee, Ming-Jer Tang, Yu-Chun Lin.

  Microtubules tightly regulate various cellular activities. Our understanding of microtubules is largely based on experiments using microtubule-targeting agents, which, however, are insufficient to dissect the dynamic mechanisms of specific microtubule populations, due to their slow effects on the entire pool of microtubules. To overcome this technological limitation, we have used chemo and optogenetics to disassemble specific microtubule subtypes, including tyrosinated microtubules, primary cilia, mitotic spindles, and intercellular bridges, by rapidly recruiting engineered microtubule-cleaving enzymes onto target microtubules in a reversible manner. Using this approach, we show that acute microtubule disassembly swiftly halts vesicular trafficking and lysosomal dynamics. It also immediately triggers Golgi and ER reorganization and slows the fusion/fission of mitochondria without affecting mitochondrial membrane potential. In addition, cell rigidity is increased after microtubule disruption owing to increased contractile stress fibers. Microtubule disruption furthermore prevents cell division, but does not cause cell death during interphase. Overall, the reported tools facilitate detailed analysis of how microtubules precisely regulate cellular architecture and functions.

Keywords:  intercellular bridge; microtubule; mitotic spindle; primary cilium; spastin

DOI:  https://doi.org/10.15252/embj.2021110472
Am J Physiol Cell Physiol. 2022 Jun 08.

Polycystin-2 (PC2) is a key determinant of in-vitro myogenesis.

Karla M Marquez-Nogueras, Virdjinija Vuchkovska, Elisabeth DiNello, Sara Osorio-Valencia, Ivana Y Kuo.

  The development of skeletal muscle (myogenesis) is a well-orchestrated process where myoblasts withdraw from the cell cycle and differentiate into myotubes. Signaling by fluxes in intracellular Ca2+ is known to contribute to myogenesis and increased mitochondria biogenesis is required to meet the metabolic demand of mature myotubes. However, gaps remain in the understanding of how intracellular Ca2+ signals can govern myogenesis. Polycystin-2 (PC2 or TRPP1) is a non-selective cation channel permeable to Ca2+. It can interact with intracellular calcium channels to control Ca2+ release, and concurrently modulates mitochondrial function and remodeling. Due to these features, we hypothesized that PC2 is a central protein in mediating both the intracellular Ca2+ responses and mitochondrial changes seen in myogenesis. To test this hypothesis, we created CRISPR/Cas9 knockout C2C12 murine myoblast cell lines. PC2 KO cells were unable to differentiate into myotubes, had impaired spontaneous Ca2+ oscillations and did not develop depolarization-evoked Ca2+ transients. The autophagic-associated pathway beclin-1 was downregulated in PC2 KO cells, and direct activation of the autophagic pathway resulted in decreased mitochondrial remodeling. Re-expression of full length PC2, but not a calcium channel dead pathologic mutant, restored the differentiation phenotype and increased the expression of mitochondrial proteins. Our results establish that PC2 is a novel regulator of in-vitro myogenesis by integrating PC2 dependent Ca2+ signals and metabolic pathways.

Keywords:  Myogenesis; PC2; TRP channels; calcium signaling; muscle differentiation

DOI:  https://doi.org/10.1152/ajpcell.00159.2021
Physiol Rep. 2022 Jun;10(11): e15349

Quantifying cilia beat frequency using high-speed video microscopy: Assessing frame rate requirements when imaging different ciliated tissues.

Luke Scopulovic, Deanne Francis, Elvis Pandzic, Richard Francis.

  Motile cilia are found in numerous locations throughout our body and play a critical role in various physiological processes. The most commonly used method to assess cilia motility is to quantify cilia beat frequency (CBF) via video microscopy. However, a large heterogeneity exists within published literature regarding the framerate used to image cilia motility for calculating CBF. The aim of this study was to determine the optimal frame rate required to image cilia motility for CBF assessment, and if the Nyquist theorem may be used to set this rate. One-second movies of cilia were collected at >600 fps from mouse airways and ependyma at room-temperature or 37°C. Movies were then down-sampled to 30-300 fps. CBF was quantified for identical cilia at different framerates by either manual counting or automated MATLAB script. Airway CBF was significantly impaired in 30 fps movies, while ependymal CBF was significantly impaired in both 60 and 30 fps movies. Pairwise comparison showed that video framerate should be at least 150 fps to accurately measure CBF, with minimal improvement in CBF accuracy in movies >150 fps. The automated script was also found to be less accurate for measuring CBF in lower fps movies than manual counting, however, this difference disappeared in higher framerate movies (>150 fps). In conclusion, our data suggest the Nyquist theorem is unreliable for setting sampling rate for CBF measurement. Instead, sampling rate should be 3-4 times faster than CBF for accurate CBF assessment. Especially if CBF calculation is to be automated.

Keywords:  cilia; cilia beat frequency; ependyma; respiratory epithelium

DOI:  https://doi.org/10.14814/phy2.15349