Regulation of dynamin oligomerization in cells: the role of dynamin-actin interactions and its GTPase activity.

Dynamin is a 96-kDa protein that has multiple oligomerization states that influence its GTPase activity. A number of different dynamin effectors, including lipids, actin filaments, and SH3-domain-containing proteins, have been implicated in the regulation of dynamin oligomerization, though their roles in influencing dynamin oligomerization have been studied predominantly in vitro using recombinant proteins. Here, we identify higher order dynamin oligomers such as rings and helices in vitro and in live cells using fluorescence lifetime imaging microscopy (FLIM). FLIM detected GTP- and actin-dependent dynamin oligomerization at distinct cellular sites, including the cell membrane and transition zones where cortical actin transitions into stress fibers. Our study identifies a major role for direct dynamin-actin interactions and dynamin's GTPase activity in the regulation of dynamin oligomerization in cells.

Dynamin rings: not just for fission.

The GTPase dynamin has captivated researchers for over two decades, even managing to establish its own research field. Dynamin's allure is partly due to its unusual biochemical properties as well as its essential role in multiple cellular processes, which include the regulation of clathrin-mediated endocytosis and of actin cytoskeleton. On the basis of the classic model, dynamin oligomerization into higher order oligomers such as rings and helices directly executes the final fission reaction in endocytosis, which results in the generation of clathrin-coated vesicles. Dynamin's role in the regulation of actin cytoskeleton is mostly explained by its interactions with a number of actin-binding and -regulating proteins; however, the molecular mechanism of dynamin's action continues to elude us. Recent insights into the mechanism and role of dynamin oligomerization in the regulation of actin polymerization point to a novel role for dynamin oligomerization in the cell.

Matrix stiffness regulates endothelial cell proliferation through septin 9.

Endothelial proliferation, which is an important process in vascular homeostasis, can be regulated by the extracellular microenvironment. In this study we demonstrated that proliferation of endothelial cells (ECs) was enhanced on hydrogels with high stiffness (HSG, 21.5 kPa) in comparison to those with low stiffness (LSG, 1.72 kPa). ECs on HSG showed markedly prominent stress fibers and a higher RhoA activity than ECs on LSG. Blockade of RhoA attenuated stress fiber formation and proliferation of ECs on HSG, but had little effect on ECs on LSG; enhancement of RhoA had opposite effects. The phosphorylations of Src and Vav2, which are positive RhoA upstream effectors, were higher in ECs on HSG. The inhibition of Src/Vav2 attenuated the HSG-mediated RhoA activation and EC proliferation but exhibited nominal effects on ECs on LSG. Septin 9 (SEPT9), the negative upstream effector for RhoA, was significantly higher in ECs on LSG. The inhibition of SEPT9 increased RhoA activation, Src/Vav2 phosphorylations, and EC proliferation on LSG, but showed minor effects on ECs on HSG. We further demonstrated that the inactivation of integrin α(v)ß(3) caused an increase of SEPT9 expression in ECs on HSG to attenuate Src/Vav2 phosphorylations and inhibit RhoA-dependent EC proliferation. These results demonstrate that the SEPT9/Src/Vav2/RhoA pathway constitutes an important molecular mechanism for the mechanical regulation of EC proliferation.

Descemet stripping automated endothelial keratoplasty using donor corneas with previous laser in situ keratomileusis or photorefractive keratectomy: a case series and donor cap histopathology.

PURPOSE: To report outcomes in Descemet stripping automated endothelial keratoplasty (DSAEK) using donor tissue from eyes that have had previous refractive surgery and to report histopathology of the donor free cap. METHODS: Retrospective case series. Preoperative and postoperative data were collected on each patient, and donor information was collected. Histopathologic evaluation was carried out on donor caps to determine the stability of the laser in situ keratomileusis (LASIK) flap and smoothness of the microkeratome cut during preparation of the donor. RESULTS: DSAEK was performed on 7 eyes using donors that had undergone photorefractive keratectomy (PRK) or LASIK. One patient received PRK donor tissue, had no interface haze, and had improvement in vision. All but one patient who received LASIK donor tissue had improvement in vision, and that patient had severe graft folds noted intraoperatively that persisted. Histopathologic examination of 3 donor caps showed mild to moderate dehiscence of the LASIK flap, but the cut interface was consistent with normal donor tissue preparation histopathology. CONCLUSIONS: We report the first case of DSAEK using PRK donor tissue, which was successful. Our experience with LASIK donor tissue was comparable to nonrefractive donor tissue with the exception of the persistent donor macrofolds in one patient.

Prospective, contralateral comparison of 120-µm and 90-µm LASIK flaps using the IntraLase FS60 femtosecond laser.

PURPOSE: To compare differences in visual acuity, contrast sensitivity, higher order ocular aberrations, quality of life, and patient-reported outcomes at 3 and 6 months postoperatively in eyes with stable myopia undergoing thin-flap (intended flap thicknesses of 120 or 90 µm) LASIK using the VISX Star S4 CustomVue excimer laser (VISX Inc), with flaps created by the IntraLase FS60 femtosecond laser (Abbott Medical Optics). METHODS: In this prospective study, thin-flap LASIK was performed contralaterally on 94 eyes: 47 eyes with 120-µm intended flap thickness and 47 eyes with 90-µm intended flap thickness. Primary outcome measures were uncorrected distance visual acuity (UDVA), corrected distance visual acuity (CDVA), contrast sensitivity, and higher order aberrations. RESULTS: At 6 months, mean values for UDVA (logMAR) were -0.064±0.077 and -0.051±0.070 in the 120-µm and 90-µm groups, respectively (n=40, P=.431). Visual acuity of 20/20 was achieved in 98% of eyes with 120-µm flaps and 95% of eyes with 90-µm flaps, whereas 20/15 vision was achieved in 50% of eyes with 120-µm flaps and 45% of eyes with 90-µm flaps (P≥.454). Both groups had significant increases in total higher order aberrations (P≤.003). Significant differences were not found between groups in contrast sensitivity (P≥.258), CDVA (P≥.726), total higher order aberrations (P≥.477), or patient-reported outcomes (P≥.132). Patients in both groups reported increased quality of life postoperatively (P≤.002). CONCLUSIONS: Under well-controlled surgical conditions, thin-flap LASIK achieved similar results in visual acuity, contrast sensitivity, and low induction of higher order aberrations in eyes with intended flap thicknesses of either 120 or 90 µm.

Programmable in situ amplification for multiplexed imaging of mRNA expression.

In situ hybridization methods enable the mapping of mRNA expression within intact biological samples. With current approaches, it is challenging to simultaneously map multiple target mRNAs within whole-mount vertebrate embryos, representing a significant limitation in attempting to study interacting regulatory elements in systems most relevant to human development and disease. Here, we report a multiplexed fluorescent in situ hybridization method based on orthogonal amplification with hybridization chain reactions (HCR). With this approach, RNA probes complementary to mRNA targets trigger chain reactions in which fluorophore-labeled RNA hairpins self-assemble into tethered fluorescent amplification polymers. The programmability and sequence specificity of these amplification cascades enable multiple HCR amplifiers to operate orthogonally at the same time in the same sample. Robust performance is achieved when imaging five target mRNAs simultaneously in fixed whole-mount and sectioned zebrafish embryos. HCR amplifiers exhibit deep sample penetration, high signal-to-background ratios and sharp signal localization.

Laser in situ keratomileusis flap complications using mechanical microkeratome versus femtosecond laser: retrospective comparison.

PURPOSE: To compare the incidence of flap complications after creation of laser in situ keratomileusis (LASIK) flaps using a zero-compression microkeratome or a femtosecond laser. SETTING: John A. Moran Eye Center, Department of Ophthalmology, University of Utah, Salt Lake City, Utah, USA. DESIGN: Evidence-based manuscript. METHODS: The flap complication rate was evaluated during the initial 18 months of experience using a zero-compression microkeratome (Hansatome) or a femtosecond laser (IntraLase FS60) for flap creation. RESULTS: The flap complication rate was 14.2% in the microkeratome group and 15.2% in the femtosecond laser group (P = .5437). The intraoperative flap complication rate was 5.3% and 2.9%, respectively (P = .0111), and the postoperative flap complication rate, 8.9% and 12.3%, respectively (P = .0201). The most common intraoperative complication in the microkeratome group was major epithelial defect/sloughing; the rate (2.6%) was statistically significantly higher than in the femtosecond laser group (P = .0006). The most common postoperative complication in both groups was diffuse lamellar keratitis (DLK) (6.0%, microkeratome; 10.6%, femtosecond laser) (P = .0002). CONCLUSION: Although the total complication rates between the 2 groups were similar, the microkeratome group had significantly more epithelial defects intraoperatively and the femtosecond laser group had significantly more DLK cases postoperatively.

Visual outcomes after wavefront-guided photorefractive keratectomy and wavefront-guided laser in situ keratomileusis: Prospective comparison.

PURPOSE: To compare visual outcomes between wavefront-guided photorefractive keratectomy (PRK) and wavefront-guided laser in situ keratomileusis (LASIK). SETTING: Academic center, Salt Lake City, Utah, USA. METHODS: In this randomized prospective study, myopic eyes were treated with wavefront-guided PRK and or wavefront-guided LASIK using a Visx Star S4 CustomVue platform with iris registration. Primary outcome measures were uncorrected (UDVA) and corrected (CDVA) distance visual acuities and manifest refraction. Secondary outcome measures were higher-order aberrations (HOAs) and contrast sensitivity. RESULTS: The PRK group comprised 101 eyes and the LASIK group, 102 eyes. At 6 months, the mean UDVA was -0.03 logMAR +/- 0.10 [SD] (20/19) and 0.07 +/- 0.09 logMAR (20/24), respectively (P = .544). In both groups, 75% eyes achieved a UDVA of 20/20 or better (P = .923); 77% of eyes in the PRK group and 88% in the LASIK group were within +/-0.50 diopter of emmetropia (P = .760). There was no statistically significant difference between groups in contrast sensitivity at 3, 6, 12, or 18 cycles per degree. The mean postoperative HOA root mean square was 0.45 +/- 0.13 mum in the PRK group and 0.59 +/- 0.22 mum in the LASIK group (P = .012), representing an increase factor of 1.22 and 1.74, respectively. CONCLUSIONS: Wavefront-guided PRK and wavefront-guided LASIK had similar efficacy, predictability, safety, and contrast sensitivity; however, wavefront-guided PRK induced statistically fewer HOAs than wavefront-guided LASIK at 6 months. FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.

Iris-fixated phakic intraocular lens implantation for correction of high myopia in microspherophakia.

We report the refractive correction of high myopia in a 23-year-old patient with idiopathic microspherophakia using iris-fixated phakic intraocular lenses (pIOLs) (Verisyse/Artisan). Four years after bilateral implantation, the uncorrected distance visual acuity was 20/25 with a correction of 20/20(-1) in both eyes. No intraoperative or postoperative complications occurred. Iris-fixated pIOLs are not recommended for every patient with microspherophakia. However, this procedure may be an option in microspherophakic patients with appropriate anterior chamber depth and no history of lens dislocation who are likely to comply with annual eye examinations. Follow-up should include monitoring the endothelial cell count and biomicroscopy for adequate space between the pIOL, the natural crystalline lens, and the corneal endothelium. Scheimpflug photography can be a valuable tool in such cases.

Cataract surgery following phakic intraocular lens implantation.

PURPOSE OF REVIEW: To review the most recent literature regarding the incidence and pathophysiology of phakic intraocular lens (pIOL) associated cataracts, surgical issues and outcomes of combined pIOL explantation/cataract surgery, and the prevention of cataract formation secondary to pIOLs. RECENT FINDINGS: The overall rate of cataracts secondary to pIOLs is low, but a disproportionate number is associated with posterior chamber pIOLs. All combination pIOL explantation/cataract surgeries resulted in the successful implantation of a posterior chamber intraocular lens in the capsular bag. We present several theories regarding the pathophysiology of anterior subcapsular cataracts secondary to posterior chamber pIOLs. In addition, we present general strategies in performing combination pIOL explantation/cataract surgery. Several methods of preoperative assessment show promise in helping prevent cataracts secondary to pIOLs, including new ultrasound methods for sulcus imaging and preoperative simulations. SUMMARY: Although the incidence of cataracts secondary to pIOLs is low, more studies regarding the pathophysiology of this phenomenon and improvement of preoperative assessment are needed, especially for posterior chamber pIOLs.

Selective adapter recruitment and differential signaling networks by VEGF vs. shear stress.

Vascular endothelial cells are continuously exposed to mechanical and chemical stimuli, such as shear stress and VEGF, respectively. It is still not clear how cells perceive these stimuli and orchestrate their responses. Studying the molecular mechanism by which shear stress and VEGF regulate the signaling pathways in bovine endothelial aortic cells, we found that VEGF induced a rapid association of VEGF receptor 2 (Flk-1) with Nck beta, but shear stress did not have such an effect. SU1498 (a specific inhibitor of Flk-1) and Nck beta(nm) (a negative mutant of Nck beta) blocked the VEGF-induced ERK and JNK activities. Only SU1498, but not Nck beta(nm), inhibited the shear-induced ERK activity. Furthermore, neither SU1498 nor Nck beta(nm) had significant effects on the shear-induced JNK activity, which can be blocked by inhibitors of Src family kinase and ROCK kinase. Therefore, mechanical (shear stress) and chemical (VEGF) stimuli diverge at the receptor Flk-1 in terms of the recruitment of the adapter protein Nck beta, and they employ different components of the complex signaling network in regulating downstream molecules, e.g., ERK and JNK.

Shear stress and VEGF activate IKK via the Flk-1/Cbl/Akt signaling pathway.

Vascular endothelial cells are continuously exposed to mechanical (e.g., shear stress) and chemical (e.g., growth factors) stimuli. It is important to elucidate the mechanisms by which cells perceive and integrate these different stimuli to regulate the downstream signaling pathways. We (50) have previously reported the shear-induced interplay between two membrane receptors, integrins and Flk-1. In the present study, we investigated the molecular mechanisms regulating the downstream IkappaB kinase (IKK) pathway in response to shear stress and VEGF. Both shear stress and VEGF induced a transient increase of IKK activity. These effects were inhibited by SU-1498, a specific Flk-1 inhibitor, and by a negative mutant of Casitas B-lineage lymphoma (Cbl) with tyrosine-to-phenylalanine mutations at sites 700, 731, and 774 (Cbl(nm)). Because Flk-1 and Cbl form a complex upon shearing or VEGF applications (50), these results suggest that shear stress and VEGF activate IKK via the receptor Flk-1 and its recruitment of the adapter protein Cbl. The inhibition of the shear- and VEGF-induced IKK activities by a negative mutant of Akt indicates that Akt acts upstream to IKK in response to shear stress and VEGF. Furthermore, SU-1498 and Cbl(-nm) abolished the shear- and VEGF-induced Akt activity, indicating that Akt acts at a level downstream to Flk-1 and Cbl. Therefore, our results indicate that the signaling events induced by shear stress and VEGF converge at the membrane receptor Flk-1 and that these stimuli share the Flk-1/Cbl/Akt pathway in activating IKK activation.