Ryan McAllister

A new chemotaxis assay shows the extreme sensitivity of axons to molecular gradients

Axonal chemotaxis is believed to be important in wiring up the developing and regenerating nervous system, but little is known about how axons actually respond to molecular gradients. We report a new quantitative assay that allows the long-term response of axons to gradients of known and controllable shape to be examined in a three-dimensional gel. Using this assay, we show that axons may be natures most-sensitive gradient detectors, but this sensitivity exists only within a narrow range of ligand concentrations. This assay should also be applicable to other biological processes that are controlled by molecular gradients, such as cell migration and morphogenesis.

Male circumcision is not the HIV ‘vaccine’ we have been waiting for!

Over the past several months some researchers and health organizations have proclaimed circumcision to be a compelling and important new HIV tool. A recent commentary claims that circumcision is “at least as good as the HIV vaccine we have been waiting for praying for and hoping to see in our lifetimes”. Thousands of African men now line up to get circumcised in the mistaken belief that it will save them from HIV as some developing nations -lacking even rudimentary medical care and clean drinking water -rush to implement mass circumcision programs with encouragement and millions of pledged dollars from the US government. In addition there are calls for implementing mass neonatal circumcision. The push to institute mass circumcision in Africa following the three randomized clinical trials (RCTs) conducted in Africa is based on an incomplete evaluation of real-world preventive effects over the long-term -effects that may be quite different outside the research setting and circumstances with their access to resources sanitary standards and intensive counseling. Moreover proposals for mass circumcision lack a thorough and objective consideration of costs in relation to hoped-for benefits. No field-test has been performed to evaluate the effectiveness complications personnel requirements costs and practicality of proposed approaches in real-life conditions. These are the classic distinctions between efficacy and effectiveness trials and between internal validity and external validity. Campaigns to promote safe-sex behaviors have been shown to accomplish a high rate of infection reduction without the surgical risks and complications of circumcision and at a much lower cost. For the health community to rush to recommend a program based on incomplete evidence is both premature and ill-advised. It misleads the public by promoting false hope from uncertain conclusions and might ultimately aggravate the problem by altering people’s behavioral patterns and exposing them and their partners to new or expanded risks. Given these problems circumcision of adults and especially of children by coercion or by false hope raises human rights concerns.

Optical neuronal guidance in three-dimensional matrices.

We demonstrate effective guidance of neurites extending from PC12 cells in a three-dimensional collagen matrix using a focused infrared laser. Processes can be redirected in an arbitrarily chosen direction in the imaging plane in approximately 30 min with an 80% success rate. In addition, the application of the laser beam significantly increases the rate of neurite outgrowth. These results extend previous observations on 2D coated glass coverslips. We find that the morphology of growth cones is very different in 3D than in 2D, and that this difference suggests that the filopodia play a key role in optical guidance. This powerful, flexible, non-contact guidance technique has potentially broad applications in tissues and engineered environments.

A Novel 3D Fibril Force Assay Implicates Src in Tumor Cell Force Generation in Collagen Networks

New insight into the biomechanics of cancer cell motility in 3D extracellular matrix (ECM) environments would significantly enhance our understanding of aggressive cancers and help identify new targets for intervention. While several methods for measuring the forces involved in cell-matrix interactions have been developed, previous to this study none have been able to measure forces in a fibrillar environment. We have developed a novel assay for simultaneously measuring cell mechanotransduction and motility in 3D fibrillar environments. The assay consists of a controlled-density fibrillar collagen gel atop a controlled-stiffness polyacrylamide (PAA) surface. Forces generated by living cells and their migration in the 3D collagen gel were measured with the 3D motion of tracer beads within the PAA layer. Here, this 3D fibril force assay is used to study the role of the invasion-associated protein kinase Src in mechanotransduction and motility. Src expression and activation are linked with proliferation, invasion, and metastasis, and have been shown to be required in 2D for invadopodia membranes to direct and mediate invasion. Breast cancer cell line MDA-MD-231 was stably transfected with GFP-tagged constitutively active Src or wild-type Src. In 3D fibrillar collagen matrices we found that, relative to wild-type Src, constitutively active Src: 1) increased the strength of cell-induced forces on the ECM, 2) did not significantly change migration speed, and 3) increased both the duration and the length, but not the number, of long membrane protrusions. Taken together, these results support the hypothesis that Src controls invasion by controlling the ability of the cell to form long lasting cellular protrusions to enable penetration through tissue barriers, in addition to its role in promoting invadopodia matrix-degrading activity.

Design and optimization of a high-speed, high- sensitivity, spinning disk confocal microscopy system

We describe the principles, design, and systems integration of a flexible, high-speed, high-sensitivity, high-resolution confocal spinning disk microscopy (SDCM) system. We present several artifacts unique to high-speed SDCM along with techniques to minimize them. We show example experimental results from a specific implementation capable of generating 3-D image stacks containing 30 2-D slices at 30 stacks per second. This implementation also includes optics for differential interference contrast (DIC), phase, and bright-field imaging, as well as an optical trap with sensitive force and position measurement.

MEDICAID COVERAGE OF CIRCUMCISION SPREADS HARM TO THE POOR

According to data reported by Leibowitz et al., lack of Medicaid coverage (and, presumably, private insurance) results in lowered circumcision rates.1 We disagree with the authors’ interpretation of these findings and with their concern that poor babies could be deprived of benefits from circumcision. On the contrary, neonatal circumcision places boys at immediate risk for complications,2 methicillin-resistant Staphylococcus aureus,3 and even death.4 Leibowitz et al. should have concluded that poor children are now at lower risk of neonatal circumcision harm. Further, as their data show, it is clearly not just poor children who are not being circumcised. In some US regions, a majority of male babies from all income brackets do not undergo circumcision. Although there is no evidence that boys not circumcised at birth are any less healthy than those who are circumcised, there is evidence of the opposite. For example, the HIV rate in America is far higher than in Europe, where males are rarely circumcised.5 The penile cancer rate is no lower in America than it is in Europe.6 A recent study showed that urinary tract infections occurred in 6 of 24 infants circumcised by a physician (25%) and in 42 of 87 infants circumcised by a religious circumciser; the calculated odds ratio for contracting a urinary tract infection were 2.8 (95% confidence interval = 1, 9.4).7 A comprehensive cost–utility study found that neonatal circumcisions complications and consequences increased health-care costs 742% beyond the cost of the circumcision itself and therefore is not a justifiable public health measure.8 It concludes that if neonatal circumcision were “cost-free, pain-free, and had no immediate complications, it was still more costly than not circumcising.”8(p584) Leibowitz et al. reinforce the overly confident notion, created by the extensive media coverage of 3 randomized clinical trials in Africa, that circumcision is partially effective against HIV. In doing so, they ignore both contradictory evidence and the fact that the trial circumstances are not generalizable to Africa, let alone America.9 Even if male circumcision were somewhat effective in reducing HIV infection among heterosexual adults in certain areas of high HIV prevalence, the leap to recommending population-wide neonatal circumcision in the United States is still unjustifiable.10 With nearly 50 million Americans lacking health insurance, and poor children going without many basic services, it is ethically, morally, and perhaps legally inappropriate that any Medicaid program continues to fund an elective and harmful procedure. We applaud the 16 states that have recognized that taxpayers should not be spending money on this unnecessary procedure and the other states that are considering dropping Medicaid coverage. No state should be wasting money on infant circumcision.

Traction force and tension fluctuations in growing axons

Actively generated mechanical forces play a central role in axon growth and guidance, but the mechanisms that underly force generation and regulation in growing axons remain poorly understood. We report measurements of the dynamics of traction stresses from growth cones of actively advancing axons from postnatal rat DRG neurons. By tracking the movement of the growth cone and analyzing the traction stress field from a reference frame that moves with it, we are able to show that there is a clear and consistent average stress field that underlies the complex spatial stresses present at any one time. The average stress field has strong maxima on the sides of the growth cone, directed inward toward the growth cone neck. This pattern represents a contractile stress contained within the growth cone, and a net force that is balanced by the axon tension. Using high time-resolution measurements of the growth cone traction stresses, we show that the stress field is composed of fluctuating local stress peaks, with a large number peaks that live for a short time, a population of peaks whose lifetime distribution follows an exponential decay, and a small number of very long-lived peaks. We show that the high time-resolution data also reveal that the tension appears to vary randomly over short time scales, roughly consistent with the lifetime of the stress peaks, suggesting that the tension fluctuations originate from stochastic adhesion dynamics.

Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy

Live-cell imaging is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT1aR) and intracellular compartments in transfected human embryonic kidney-293 (HEK) cells following stimulation with angiotensin II (Ang II). HEK cells are transiently transfected with plasmid DNA containing AT1aR tagged with enhanced green fluorescent protein (EGFP). Lysosomes are identified with a red fluorescent dye. Live-cell images are captured on a laser scanning confocal microscope after Ang II stimulation and analyzed by software in three dimensions (3D, voxels) over time. Live-cell imaging enables investigations into receptor trafficking and avoids confounds associated with fixation, and in particular, the loss or artefactual displacement of EGFP-tagged membrane receptors. Thus, as individual cells are tracked through time, the subcellular localization of receptors can be imaged and measured. Images must be acquired sufficiently rapidly to capture rapid vesicle movement. Yet, at faster imaging speeds, the number of photons collected is reduced. Compromises must also be made in the selection of imaging parameters like voxel size in order to gain imaging speed. Significant applications of live-cell imaging are to study protein trafficking, migration, proliferation, cell cycle, apoptosis, autophagy and protein-protein interaction and dynamics, to name but a few.