Dynamics Movement and Protein Composition of Ciliary Bulb in a Primary Cilium
Dynamics movement and protein composition of ciliary bulb in a primary cilium
Protein composition and movements of membrane swellings associated with primary cilia
Ashraf M. Mohieldin1,2, Hanan S. Haymour2, Shao T. Lo2, Wissam A. AbouAlaiwi2, Kimberly F. Atkinson3, Christopher J. Ward4, Min Gao5, Oliver Wessely6, Surya M. Nauli1,2,3
1Department of Medicinal & Biological Chemistry, University of Toledo, Health Science Building, Toledo, OH 43614.
2Department of Pharmacology & Experimental Therapeutics, University of Toledo, Health Science Building, Toledo, OH 43614.
3Department of Biomedical & Pharmaceutical Sciences, Chapman University, Irvine, CA 92618.
4Department of Medicine, The Kidney Institute, University of Kansas Medical Center, Kansas City, KS 66160.
5Liquid Crystal Institute, Kent State University, 1425 University Esplanade, Kent, OH 44242.
6Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195.
Corresponding:
Surya Nauli
Chapman University
Harry and Diane Rinker Health Science Campus
9401 Jeronimo Road.
Irvine, CA 92618-1908
Tel: 714-516-5485
Email: ;
Co-corresponding:
WissamAbouAlaiwi
The University of Toledo
3000 Arlington Ave
Toledo, OH 43614
Tel: 419-383-1949
Email:
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Supplemental Figures
Supp Figure 1. Stable Bicc-1, St3gal5 and Pkd2 knockdown cell lines were generated.
The shRNA-GFP knockdown efficiency in LLCPK cells was verified with flow cytometer. Stable Bicc-1, St3gal5 and Pkd2 cell lines showed a high knockdown efficiency as depicted by GFP signal intensity (M3 gated channel).
Supp Figure 2. Cilarybulbmembrane swelling was detected in mouse vascular endothelial cells.
Both standard (a) and formvar (b) techniques were used to show the presence of ciliary bulb in relatively short endothelial cilia. Acetylated-α-tubulin (green) was used as ciliary marker, whereas pericentrin (red) and DAPI (blue) were used to as centrosomal and nuclear markers, respectively. Arrows indicate the presence of ciliary bulbs at the tip of cilia. Bar=3 m.
Supp Figure 3. Ciliarybulbmembrane swelling was differentially positioned in static and flow conditions.
Most bulbsswellings were positioned around the mid of ciliary shaft in static no-flow condition, but they tended to move to the tip of cilia under fluid-shear stress (Figure 2). In about 10% of cases, however, the bulb would move to the tip of a cilium momentarily before move back along the ciliary shaft (a). Furthermore, in a very rare occasion the bulb could maintain at the bottom of a cilium under fluid-fluid flow (b).
Supp Figure 4. Flow-induced cilarybulbswelling formation was magnitude- and time-independent events.
To examine if the magnitude of shear stress would induce bulbswelling formation differentially, we challenged the cells at low (10.3±2.1L/min), medium (21.6±2.4L/min) or high(35.3±1.1L/min) flow rate. Because the appearance of the bulbswelling was so random (time-independent), we calculated those cells that responded to flow-induced bulb formation within a minute. All in all, flow-induced bulb formation was observed in about 80-90% of the cells. For those cells that responded to fluid-shear stress within a minute, there was no indication that magnitude of fluid-shear stress would differentially affect bulb formation (magnitude-independent).
Supp Figure 5. Ciliarybulb membrane swelling was detected in renal epithelial cells in vitro and in vivo, and it contained integral proteins.
Scanning electron microscope revealed the absence (left panel) and presence (right panel) of ciliarybulbswelling in LLCPK cells (a). The ciliarybulbswelling was also seen in mouse renal epithelia (b). Two representative images were independently taken showing that the ciliarybulbswelling was an integral structure of the ciliary membrane. Further studies with freeze fracture transmission microscopy (FFTM) further demonstrated the presence of ciliarybulbswelling at the tip of cilium with residual integral proteins on the cilia and ciliarybulbswelling region (c). Right panel shows an enlargement image from the left image. All primary cilia were pseudocolored with green, and white arrows point to ciliary bulbs.
Supplemental Movies
Movie 1. Movement of ciliarybulbmembrane swelling under static condition
Primary cilium of an LLCPK renal epithelium showed the dynamic movement of ciliarybulbswelling oscillating up and down along the ciliary shaft. The ciliarybulbswelling never reached to the tip of the cilium. The black arrow points at the ciliarybulbswelling. Time is shown in minute:second.
Movie 2. The additional appearance of new ciliary bulb during flow-shear stress.
Primary cilium of an LLCPK renal epithelium showed the sensitivity of ciliarybulbswelling in response to mechanical stimulus. Asmall pulse of fluid flow, enough to generate a small movement on the cilium,induced the appearance of another bulb (red arrow) along with the preexisting bulb (black arrow). Both ciliarybulbsswellingsmoved to the tip of the cilia during the course of the fluid-flow, but they returned to move dynamically along the ciliary shaft when fluid-flowwas ceased. Time is shown in minute:second.
Movie 3. The appearance of ciliarybulbmembrane swelling from the middle of the ciliary shaft.
Ciliarybulbswelling appearance could be induced by fluid-flow from the middle of ciliary shaft (red arrow) in an LLCPK renal epithelium. Time is shown in minute:second.
Supplemental Methods
Flow cytometer
All stable cell line St3gal5-GFP, Bicc1-GFP and Pkd2-GFP were further analyzed for GFP expression efficiency. Prior to analysis, cells were rinsed with 1X PBS, detached with trypsin, pelleted at 4000 rpm, resuspended in PBS and analyzed using C6 Flow Cytometer (Accuri Cytometers, MI, USA).
Scanning electron microscopy (SEM)
Briefly, cells were fixed with 2.5% of glutaraldehyde in 0.085 M cacodylate buffer for 1 hour and then washed with cacodylate buffer for 5 minutes. A 1% of osmium tetroxide in 0.085 M cacodylate buffer was used for 10 minutes and dehydrated the samples through a graded series of ethanol. The sample was then infiltrated with hexamethyldisilazane and left air dry overnight. The sample was then coated with gold particles and examined (Hitachi S-4800).
Freeze Fracture Transmission Electron Microscopy (FFTEM)
Sample was prepared on replica specimens prepared by combining high pressure freezing and freeze fracture. LLCPK cells were grown directly inside gold-plated FF carriers, and they were thencryo-fixed with the original culture media in a Leica EM Pact 2 high-pressure freezer. To optimize sample preparation, we also replaced the culture media in some of the carriers with 20% dextran solution (40K Mr) right before the freezing, which did not result in noticeable difference. After high pressure freezing, the carriers were quickly transferred into a freeze-fracture vacuum chamber (BalTec BAF060) and fractured at -165°C. The topography of the fractured surface was then replicated by the deposition of a thin (~4 nm) Pt/C layer at 45° followed by a continuous carbon film (~20 nm) deposited normally to the surface. The replicas were collected by holey lacey carbon grids after dissolving all the culture media with water, and investigated in a FEI Tecnai F20 TEM operated at 200 KV.
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