Arp2/3 complex inhibition radically alters lamellipodial actin architecture, suspended cell shape, and the cell spreading process

© The Author(s), 2015. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Molecular Biology of the Cell 26 (2015): 887-900, doi:10.1091/mbc.E14-07-1244.Recent studies have investigated the dendritic actin cytoskeleton of the cell edge's lamellipodial (LP) region by experimentally decreasing the activity of the actin filament nucleator and branch former, the Arp2/3 complex. Here we extend these studies via pharmacological inhibition of the Arp2/3 complex in sea urchin coelomocytes, cells that possess an unusually broad LP region and display correspondingly exaggerated centripetal flow. Using light and electron microscopy, we demonstrate that Arp2/3 complex inhibition via the drug CK666 dramatically altered LP actin architecture, slowed centripetal flow, drove a lamellipodial-to-filopodial shape change in suspended cells, and induced a novel actin structural organization during cell spreading. A general feature of the CK666 phenotype in coelomocytes was transverse actin arcs, and arc generation was arrested by a formin inhibitor. We also demonstrate that CK666 treatment produces actin arcs in other cells with broad LP regions, namely fish keratocytes and Drosophila S2 cells. We hypothesize that the actin arcs made visible by Arp2/3 complex inhibition in coelomocytes may represent an exaggerated manifestation of the elongate mother filaments that could possibly serve as the scaffold for the production of the dendritic actin network.This research was supported by National Science Foundation STEP grant 0856704 to Dickinson College, student/faculty summer research grants from the Dickinson College Research and Development Committee, Laura and Arthur Colwin Summer Research Fellowships from the Marine Biological Laboratory to J.H.H. and C.B.S., National Institutes of Health Grant EB002583 to R.O., and National Science Foundation collaborative research grants to J.H.H. (MCB-1412688) and C.B.S. (MCB-1412734)

Osteochondral Allograft Transplantation of the Knee in Patients with an Elevated Body Mass Index.

OBJECTIVE:To characterize the graft survivorship and clinical outcomes of osteochondral allograft transplantation (OCA) of the knee in patients with an elevated body mass index (BMI). DESIGN:Prospective data on 38 consecutive patients with a BMI ≥30 kg/m2 treated with OCA from 2000 to 2015 were reviewed. Complications, reoperations, and patient responses to validated outcome measures were examined. Failures were defined by any removal/revision of the allograft or conversion to arthroplasty. RESULTS:Thirty-one knees in 31 patients (mean age, 35.4 years [range, 17-61 years]; 87% male) met the inclusion criteria. Mean BMI was 32.9 kg/m2 (range, 30-39 kg/m2). Mean chondral defect size was 6.4 cm2 (range, 1.0-15.3 cm2). Prior to OCA, 23 patients (74%) had undergone previous surgery to the ipsilateral knee. Mean duration of follow-up was 4.1 years (range, 2-11 years). After OCA, 5 knees (13%) underwent conversion to unicompartmental (1) or total (4) knee arthroplasty. Two- and 5-year graft survivorship were 87% and 83%, respectively. At final follow-up, clinically significant improvements were noted in the pain (49.3-72.6) and physical functioning (52.9-81.3) subscales of the Short Form-36 ( P ≤ 0.001), International Knee Documentation Committee subjective form (43.5-67.0; P = 0.002), Knee Outcome Survey-Activities of Daily Living (58.2-80.4; P = 0.002), and overall condition subscale of the Cincinnati Knee Rating System (4.7-6.9; P = 0.046). CONCLUSIONS:OCA can be a successful midterm treatment option for focal cartilage defects of the knee in select patients with a BMI ≥30 kg/m2

Osteochondral Allograft Transplantation of the Knee in Patients with an Elevated Body Mass Index

OBJECTIVE:To characterize the graft survivorship and clinical outcomes of osteochondral allograft transplantation (OCA) of the knee in patients with an elevated body mass index (BMI). DESIGN:Prospective data on 38 consecutive patients with a BMI ≥30 kg/m2 treated with OCA from 2000 to 2015 were reviewed. Complications, reoperations, and patient responses to validated outcome measures were examined. Failures were defined by any removal/revision of the allograft or conversion to arthroplasty. RESULTS:Thirty-one knees in 31 patients (mean age, 35.4 years [range, 17-61 years]; 87% male) met the inclusion criteria. Mean BMI was 32.9 kg/m2 (range, 30-39 kg/m2). Mean chondral defect size was 6.4 cm2 (range, 1.0-15.3 cm2). Prior to OCA, 23 patients (74%) had undergone previous surgery to the ipsilateral knee. Mean duration of follow-up was 4.1 years (range, 2-11 years). After OCA, 5 knees (13%) underwent conversion to unicompartmental (1) or total (4) knee arthroplasty. Two- and 5-year graft survivorship were 87% and 83%, respectively. At final follow-up, clinically significant improvements were noted in the pain (49.3-72.6) and physical functioning (52.9-81.3) subscales of the Short Form-36 ( P ≤ 0.001), International Knee Documentation Committee subjective form (43.5-67.0; P = 0.002), Knee Outcome Survey-Activities of Daily Living (58.2-80.4; P = 0.002), and overall condition subscale of the Cincinnati Knee Rating System (4.7-6.9; P = 0.046). CONCLUSIONS:OCA can be a successful midterm treatment option for focal cartilage defects of the knee in select patients with a BMI ≥30 kg/m2

Arp2/3 complex inhibition radically alters lamellipodial actin architecture, suspended cell shape, and the cell spreading process

Recent studies have investigated the dendritic actin cytoskeleton of the cell edge's lamellipodial (LP) region by experimentally decreasing the activity of the actin filament nucleator and branch former, the Arp2/3 complex. Here we extend these studies via pharmacological inhibition of the Arp2/3 complex in sea urchin coelomocytes, cells that possess an unusually broad LP region and display correspondingly exaggerated centripetal flow. Using light and electron microscopy, we demonstrate that Arp2/3 complex inhibition via the drug CK666 dramatically altered LP actin architecture, slowed centripetal flow, drove a lamellipodial-to-filopodial shape change in suspended cells, and induced a novel actin structural organization during cell spreading. A general feature of the CK666 phenotype in coelomocytes was transverse actin arcs, and arc generation was arrested by a formin inhibitor. We also demonstrate that CK666 treatment produces actin arcs in other cells with broad LP regions, namely fish keratocytes and Drosophila S2 cells. We hypothesize that the actin arcs made visible by Arp2/3 complex inhibition in coelomocytes may represent an exaggerated manifestation of the elongate mother filaments that could possibly serve as the scaffold for the production of the dendritic actin network