Mechanical properties of primary cilia regulate the response to fluid flow

2010 ◽  
Vol 298 (5) ◽  
pp. F1096-F1102 ◽  
Author(s):  
Susanna Rydholm ◽  
Gordon Zwartz ◽  
Jacob M. Kowalewski ◽  
Padideh Kamali-Zare ◽  
Thomas Frisk ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Fluid Flow ◽  
Calcium Signaling ◽  
Mammalian Cells ◽  
Primary Cilia ◽  
Cyst Formation ◽  
Calcium Signal ◽  
Autosomal Dominant Polycystic Kidney ◽  
Stress Buildup ◽  
Surrounding Membrane

The primary cilium is a ubiquitous organelle present on most mammalian cells. Malfunction of the organelle has been associated with various pathological disorders, many of which lead to cystic disorders in liver, pancreas, and kidney. Primary cilia have in kidney epithelial cells been observed to generate intracellular calcium in response to fluid flow, and disruption of proteins involved in this calcium signaling lead to autosomal dominant polycystic kidney disease, implying a direct connection between calcium signaling and cyst formation. It has also been shown that there is a significant lag between the onset of flow and initiation of the calcium signal. The present study focuses on the mechanics of cilium bending and the resulting calcium signal. Visualization of real-time cilium movements in response to different types of applied flow showed that the bending is fast compared with the initiation of calcium increase. Mathematical modeling of cilium and surrounding membrane was performed to deduce the relation between bending and membrane stress. The results showed a delay in stress buildup that was similar to the delay in calcium signal. Our results thus indicate that the delay in calcium response upon cilia bending is caused by mechanical properties of the cell membrane.

scholarly journals Polycystic Diseases in Visceral Organs

2011 ◽  
Vol 2011 ◽  
pp. 1-7 ◽  
Author(s):  
Shakila Abdul-Majeed ◽  
Surya M. Nauli
Keyword(s):  
Mammalian Cells ◽  
Primary Cilia ◽  
Polycystic Ovarian Syndrome ◽  
Cyst Formation ◽  
Apical Surface ◽  
Polycystic Kidney ◽  
Pancreas Disease ◽  
Polycystic Liver ◽  
And Function ◽  
Ovarian Syndrome

Primary cilia are nonmotile, microtubule-based, antenna-like organelles projecting from the apical surface of most mammalian cells. Elegant studies have established the importance of ciliary structure and function in signal transduction and the sensory roles of cilia in maintaining healthy cellular state. In particular, dysfunctional cilia have been implicated in a large number of diseases mainly characterized by the presence of fluid-filled cysts in various organs. Aside from polycystic kidney disease (PKD), however, the roles of cilia in polycystic liver disease (PLD), polycystic pancreas disease (PPD), and polycystic ovarian syndrome (PCOS) are still very vague. In addition, although gender and sex hormones are known to regulate cyst formation, their roles in regulating physiological functions of cilia need to be further explored.

scholarly journals Polycystin-2 (TRPP2) Regulates Primary Cilium Length in LLC-PK1 Renal Epithelial Cells

2020 ◽  
Author(s):  
Paula L. Perez ◽  
Noelia Scarinci ◽  
Horacio F. Cantiello ◽  
María del Rocío Cantero
Keyword(s):  
Epithelial Cells ◽  
Gene Silencing ◽  
Primary Cilium ◽  
Primary Cilia ◽  
Cyst Formation ◽  
Channel Blockers ◽  
Wild Type ◽  
Polycystic Kidney ◽  
Renal Epithelial Cells ◽  
Autosomal Dominant Polycystic Kidney

AbstractPolycystin-2 (PC2, TRPP2) is a Ca2+ permeable non-selective cation channel whose dysfunction generates autosomal dominant polycystic kidney disease (ADPKD). PC2 is present in different cell locations, including the primary cilium of renal epithelial cells. Little is known, however, as to whether PC2 contributes to the structure of the primary cilium. Here, we explored the effect(s) of external Ca2+, PC2 channel blockers, and PKD2 gene silencing on the length of primary cilia in wild type LLC-PK1 renal epithelial cells. To identify primary cilia and measure their length, confluent cell monolayers were fixed and immuno-labeled with an anti-acetylated α-tubulin antibody. Although primary cilia length measurements did not follow a Normal distribution, data were normalized by Box-Cox transformation rendering statistical difference under all experimental conditions. Cells exposed to high external Ca2+ (6.2 mM) decreased a 13.5% (p < 0.001) primary cilia length as compared to controls (1.2 mM Ca2+). In contrast, the PC2 inhibitors amiloride (200 μM) and LiCl (10 mM), both increased primary ciliary length by 33.2% (p < 0.001), and 17.4% (p < 0.001), respectively. PKD2 gene silencing by siRNA also elicited a statistically significant, 10.3% (p < 0.001) increase in primary cilia length, as compared to their respective scrambled RNA transfected cells. The data indicate that maneuvers that either regulate PC2 function or gene expression, modify the length of primary cilia in renal epithelial cells. Proper regulation of PC2 function in the primary cilium may be essential in the onset of mechanisms that trigger cyst formation in ADPKD.Significance StatementPolycystin-2 (PC2, TRPP2) is a Ca2+ permeable non-selective cation channel causing the autosomal dominant polycystic kidney disease (ADPKD). The importance of intact cilia and of fully functional polycystins in the onset of ADPKD cyst formation, point to yet unknown signaling mechanisms occurring within this organelle. We determined that the extracellular Ca2+ concentration, PC2 channel blockers, and PKD2 gene silencing, all contribute to the length of primary cilia in wild type LLC-PK1 renal epithelial cells. The data indicate that proper regulation of PC2 function in the primary cilium may be essential in the onset of mechanisms that trigger cyst formation in ADPKD.

scholarly journals The heteromeric PC-1/PC-2 polycystin complex is activated by the PC-1 N-terminus

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Kotdaji Ha ◽  
Mai Nobuhara ◽  
Qinzhe Wang ◽  
Rebecca V Walker ◽  
Feng Qian ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Primary Cilia ◽  
N Terminus ◽  
Functional Understanding ◽  
Soluble Ligand ◽  
Autosomal Dominant Polycystic Kidney ◽  
Necessary And Sufficient ◽  
Autoproteolytic Cleavage

Mutations in the polycystin proteins, PC-1 and PC-2, result in autosomal dominant polycystic kidney disease (ADPKD) and ultimately renal failure. PC-1 and PC-2 enrich on primary cilia, where they are thought to form a heteromeric ion channel complex. However, a functional understanding of the putative PC-1/PC-2 polycystin complex is lacking due to technical hurdles in reliably measuring its activity. Here we successfully reconstitute the PC-1/PC-2 complex in the plasma membrane of mammalian cells and show that it functions as an outwardly rectifying channel. Using both reconstituted and ciliary polycystin channels, we further show that a soluble fragment generated from the N-terminal extracellular domain of PC-1 functions as an intrinsic agonist that is necessary and sufficient for channel activation. We thus propose that autoproteolytic cleavage of the N-terminus of PC-1, a hotspot for ADPKD mutations, produces a soluble ligand in vivo. These findings establish a mechanistic framework for understanding the role of PC-1/PC-2 heteromers in ADPKD and suggest new therapeutic strategies that would expand upon the limited symptomatic treatments currently available for this progressive, terminal disease.

Polycystic kidney disease and renal injury repair: common pathways, fluid flow, and the function of polycystin-1

2007 ◽  
Vol 293 (5) ◽  
pp. F1423-F1432 ◽  
Author(s):  
Thomas Weimbs
Keyword(s):  
Fluid Flow ◽  
Kidney Disease ◽  
Polycystic Kidney Disease ◽  
Protein Interactions ◽  
Renal Injury ◽  
Primary Cilia ◽  
Normal Function ◽  
Polycystic Kidney ◽  
Critical Function ◽  
Autosomal Dominant Polycystic Kidney

The root cause for most cases of autosomal-dominant polycystic kidney disease (ADPKD) is mutations in the polycystin-1 (PC1) gene. While PC1 has been implicated in a perplexing variety of protein interactions and signaling pathways, what its normal function is and why its disruption leads to the proliferation of renal epithelial cells are unknown. Recent results suggest that PC1 is involved in mechanotransduction by primary cilia measuring the degree of luminal fluid flow. PC1 has also recently been shown to regulate the mTOR and signal transducers and activators of transcription (STAT) 6 pathways. These two pathways are normally dormant in the healthy kidney but are activated in response to injury and appear to drive a proliferative repair response. This review develops the idea that a critical function of PC1 and primary cilia in the adult kidney may be to sense renal injury by detecting changes in luminal fluid flow and to trigger proliferation. Constitutive activation of these pathways in ADPKD would lead to the futile attempt to repair a nonexisting injury, resulting in cyst growth. The existence of many known cellular and molecular similarities between renal repair and ADPKD supports this model.

scholarly journals Ca2+-Dependent Regulation by the Cyclic AMP Pathway of Primary Cilium Length in LLC-PK1 Renal Epithelial Cells

2020 ◽  
Author(s):  
Noelia Scarinci ◽  
Paula L. Perez ◽  
María del Rocío Cantero ◽  
Horacio F. Cantiello
Keyword(s):  
Kidney Disease ◽  
Epithelial Cells ◽  
Primary Cilium ◽  
Primary Cilia ◽  
Autosomal Dominant ◽  
Cyst Formation ◽  
Polycystic Kidney ◽  
Renal Epithelial Cells ◽  
Autosomal Dominant Polycystic Kidney ◽  
Cilium Length

AbstractThe primary cilium is a sensory organelle projecting from the apical surface of renal epithelial cells. Dysfunctional cilia have been linked to a number of genetic diseases known as ciliopathies, which include autosomal dominant polycystic kidney disease (ADPKD). Previous studies have determined that renal epithelial primary cilia express both the polycystin-2 (PC2, TRPP2) channel and the type-2 vasopressin receptor (V2R), coupled to local cAMP production. However, little is known as to how Ca2+ and cAMP signals lead to changes in the length of the primary cilium. Here, we explored how cAMP signals regulate the length of the primary cilium in wild type LLC-PK1 renal epithelial cells. Primary cilia length was determined by immunocytochemical labeling of the ciliary axoneme. Treatment of cells with the cAMP analog 8-Br-cAMP (1 mM) in normal external Ca2+ (1.2 mM) produced a 25.3% increase (p < 0.0001) in the length of the primary cilium, a phenomenon also observed in cells exposed to high external Ca2+ (6.2 mM). However, exposure of cells to vasopressin (AVP, 10 μM), which also increases cAMP in primary cilia of LLC-PK1 cells, mimicked the effect of 8-Br-cAMP in normal, but not in high Ca2+. Further, specific gene silencing of PC2 expression further increased primary cilium length after 8-Br-cAMP treatment, in normal, but not high Ca2+. The encompassed data indicate a crosstalk between the cAMP and Ca2+ signals to modulate the length of the primary cilium, in a phenomenon that implicates the expression of PC2.Significance StatementMorphological changes in primary cilia have been linked to genetic disorders, including autosomal dominant polycystic kidney disease (ADPKD), a major cause of kidney disease. Both cAMP and Ca2+ are universal second messengers that regulate polycystin-2 (PC2, TRPP2), a Ca2+ permeable non-selective cation channel implicated in ADPKD, and expressed in the primary cilium of renal epithelial cells. Despite current interest, little is known as to how second messenger systems and how aberrant regulation of PC2 may link primary cilium structure with cyst formation in ADPKD. Here we determined that both the cAMP analog 8-Br-cAMP and vasopressin increase the length of the primary cilium in renal epithelial cells. However, this phenomenon depends of external Ca2+ and PKD2 gene silencing. Proper cAMP signaling may be essential in the control of the primary cilium of renal epithelial cells, and the onset of cyst formation in ADPKD.

scholarly journals Tissue-Resident Macrophages Promote Renal Cystic Disease

2019 ◽  
Vol 30 (10) ◽  
pp. 1841-1856 ◽  
Author(s):  
Kurt A. Zimmerman ◽  
Cheng J. Song ◽  
Zhang Li ◽  
Jeremie M. Lever ◽  
David K. Crossman ◽  
...  
Keyword(s):  
Renal Injury ◽  
Primary Cilia ◽  
Kinase Inhibitor ◽  
Renal Cyst ◽  
Cyst Formation ◽  
Mutant Mice ◽  
Cystic Disease ◽  
Renal Cystic Disease ◽  
Resident Macrophage ◽  
Membrane Bound

BackgroundMutations affecting cilia proteins have an established role in renal cyst formation. In mice, the rate of cystogenesis is influenced by the age at which cilia dysfunction occurs and whether the kidney has been injured. Disruption of cilia function before postnatal day 12–14 results in rapid cyst formation; however, cyst formation is slower when cilia dysfunction is induced after postnatal day 14. Rapid cyst formation can also be induced in conditional adult cilia mutant mice by introducing renal injury. Previous studies indicate that macrophages are involved in cyst formation, however the specific role and type of macrophages responsible has not been clarified.MethodsWe analyzed resident macrophage number and subtypes during postnatal renal maturation and after renal injury in control and conditional Ift88 cilia mutant mice. We also used a pharmacological inhibitor of resident macrophage proliferation and accumulation to determine the importance of these cells during rapid cyst formation.ResultsOur data show that renal resident macrophages undergo a phenotypic switch from R2b (CD11clo) to R2a (CD11chi) during postnatal renal maturation. The timing of this switch correlates with the period in which cyst formation transitions from rapid to slow following induction of cilia dysfunction. Renal injury induces the reaccumulation of juvenile-like R2b resident macrophages in cilia mutant mice and restores rapid cystogenesis. Loss of primary cilia in injured conditional Ift88 mice results in enhanced epithelial production of membrane-bound CSF1, a cytokine that promotes resident macrophage proliferation. Inhibiting CSF1/CSF1-receptor signaling with a CSF1R kinase inhibitor reduces resident macrophage proliferation, R2b resident macrophage accumulation, and renal cyst formation in two mouse models of cystic disease.ConclusionsThese data uncover an important pathogenic role for resident macrophages during rapid cyst progression.

Revealing Mechanical Strength of Nanopores Using Atomic Finite Element Techniques

2018 ◽  
Vol 934 ◽  
pp. 24-29
Author(s):  
Prapasiri Pongprayoon ◽  
Attaphon Chaimanatsakun
Keyword(s):  
Mechanical Properties ◽  
Finite Element Method ◽  
Finite Element ◽  
Fluid Flow ◽  
Mechanical Strength ◽  
Impact Force ◽  
Pore Surface ◽  
Vertical Rectangle ◽  
The Impact ◽  
Graphene Nanopore

Graphene nanopore has been widely employed in nanofilter or nanopore devices due to its outstanding properties. The understanding of its mechanical properties at nanoscale is crucial for device improvement. In this work, the mechanical properties of graphene nanopore is thus investigated using atomistic finite element method (AFEM). Four graphene models with different pore shapes (circular (CR), horizontal rectangle (RH), and vertical rectangle (RV)) in sub-nm size which could be successfully fabricated experimentally have been studied here. The force normal to a pore surface is applied to mimic the impact force due to a fluid flow. Increasing pore size results in the reduction in its strength. Comparing among different pore shapes with comparable sizes, the order of pore strength is CR>RH>RV>SQ. In addition, we observe that the direction of pore alignment and geometries of pore edge also play a key role in mechanical strength of nanopores.

scholarly journals The retromer complex regulates C. elegans development and mammalian ciliogenesis

2021 ◽  
Author(s):  
Shuwei Xie ◽  
Ellie Smith ◽  
Carter Dierlam ◽  
Danita Mathew ◽  
Angelina Davis ◽  
...  
Keyword(s):  
Potential Function ◽  
Primary Cilium ◽  
Mammalian Cells ◽  
Primary Cilia ◽  
Vulval Development ◽  
Sorting Nexin ◽  
Capping Protein ◽  
C Elegans ◽  
Retromer Complex ◽  
Mother Centriole

The mammalian retromer is comprised of subunits VPS26, VPS29 and VPS35, and a more loosely-associated sorting nexin (SNX) heterodimer. Despite known roles for the retromer in multiple trafficking events in yeast and mammalian cells, its role in development is poorly understood, and its potential function in primary ciliogenesis remains unknown. Using CRISPR-Cas9 editing, we demonstrated that vps-26 homozygous knockout C. elegans have reduced brood sizes and impaired vulval development, as well as decreased body length which has been linked to defects in primary ciliogenesis. Since many endocytic proteins are implicated in the generation of primary cilia, we addressed whether the retromer regulates ciliogenesis in mammalian cells. We observed VPS35 localized to the primary cilium, and depletion of VPS26, VPS35 or SNX1/SNX5 led to decreased ciliogenesis. Retromer also coimmunoprecipitated with the capping protein, CP110, and was required for its removal from the mother centriole. Herein, we characterize new roles for the retromer in C. elegans development and in the regulation of ciliogenesis in mammalian cells, and suggest a novel role for the retromer in CP110 removal from the mother centriole.

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