Helen M. Buettner

Transport models for chemotactic cell populations based on individual cell behavior

A simple, generalized transport equation for the migration of chemotactic cell populations is derived from a probabilistic model in one spatial dimension. This general equation is then made specific for the cases of flagellar bacteria and polymorphonuclear leukocytes, based on quantitative information concerning chemosensory movement behavior of individual cells of these types. The resulting specialized equations allow a priori estimation of the polulation transport coefficients (μ, the random motility coefficient, and x, the chemotaxis coefficient) in terms of individual cell movement properties (such as speed, directional persistence time, and directional orientation bias). Thus, our model permits cell population migration to be related quantitatively to individual cell behavior. Examples of application of our models to experimental data are provided. Despite the simplifying approximations involved in our derivations, especially in the extrapolation to higher spatial dimensions, the models demonstrate a satisfactory and very useful ability to quantitatively interpret population assays for bacterial and leukocyte chemotactic migration.

Neurite Outgrowth is Directed by Schwann Cell Alignment in the Absence of Other Guidance Cues

Schwann cells enhance axonal regeneration following nerve injury in vivo and provide a favorable substrate for neurite outgrowth in vitro. However, much remains unknown about the nature of interactions that occur between Schwann cells and growing neurites. In this paper, we describe direct evidence of the ability of Schwann cell alignment alone to direct neurite outgrowth. Previously, we reported that laminin micropatterns can be used to align Schwann cells and thus create oriented Schwann cell monolayers. In the current study, dissociated rat spinal neurons were seeded onto oriented Schwann cell monolayers, whose alignment provided the only directional cue for growing neurites, and neurite alignment with the underlying Schwann cells was analyzed. The orientation of neurite outgrowth mimicked that of the Schwann cells. Associations observed between neurites and Schwann cells suggest that Schwann cells may guide neurite outgrowth through both topographical and molecular mechanisms. This work demonstrates that Schwann cell alignment can direct neurite outgrowth in the absence of other directional cues, and provides a new method for examining neuronal–Schwann cell interactions in vitro.

Kinetics of microtubule catastrophe assessed by probabilistic analysis

Microtubules are cytoskeletal filaments whose self-assembly occurs by abrupt switching between states of roughly constant growth and shrinkage, a process known as dynamic instability. Understanding the mechanism of dynamic instability offers potential for controlling microtubule-dependent cellular processes such as nerve growth and mitosis. The growth to shrinkage transitions (catastrophes) and the reverse transitions (rescues) that characterize microtubule dynamic instability have been assumed to be random events with first-order kinetics. By direct observation of individual microtubules in vitro and probabilistic analysis of their distribution of growth times, we found that while the slower growing and biologically inactive (minus) ends obeyed first-order catastrophe kinetics, the faster growing and biologically active (plus) ends did not. The non-first-order kinetics at plus ends imply that growing microtubule plus ends have an effective frequency of catastrophe that depends on how long the microtubules have been growing. This frequency is low initially but then rises asymptotically to a limiting value. Our results also suggest that an additional parameter, beyond the four parameters typically used to describe dynamic instability, is needed to account for the observed behavior and that changing this parameter can significantly affect the distribution of microtubule lengths at steady state.

Schwann Cell Response to Micropatterned Laminin Surfaces

In the peripheral nervous system, Schwann cells are closely associated with, and play key roles in, the development, maintenance, and regeneration of peripheral neurons. Following injury, Schwann cell orientation may also play a role in guiding regenerating axons. To aid in the investigation of these interactions between Schwann cells and growing neurites, we have developed a method of controlling Schwann cell placement and orientation in vitro by using microlithographically patterned laminin substrates, alternating 20-microm regions of laminin with bovine serum albumin (BSA) stripes. The Schwann cells predominantly attached and elongated on the laminin stripes and organized into multicellular aggregates that were oriented with the micropattern. A detailed analysis of Schwann cell aggregate orientation and shape demonstrated a strong dependence on time. At 1 h after seeding the cells, 70% of the aggregates were oriented with respect to the micropattern; 94% were oriented at 24 h. Variations in laminin concentration and seeding density were also investigated. The only significant differences in Schwann cell response occurred 1 h after seeding (the earliest time point the cultures were observed), and the main factor controlling the cellular orientation appeared to be the presence of the laminin-BSA interface. This ability to control cell orientation and placement provides a tool for future investigations of Schwann cell-neuronal interactions in vitro.

The Influence of Female Role Models on Women’s Implicit Science Cognitions

Can female science professors benefit women? Women’s negative implicit cognitions about science, technology, engineering, and mathematics (STEM) disciplines impact performance in these fields, marking implicit associations as a space for potential change to improve women’s participation in STEM. Examining college student science majors (N = 320, 63% women) enrolled in chemistry and engineering courses, our study investigates how meaningful contact with female role models impacts women’s implicit cognitions about STEM. We used the Implicit Association Test to measure attitudes toward science, identification with science, and gendered stereotypes about science, and we compared students with female versus male professors. Our study first demonstrates both direct and indirect paths between implicit cognitions and women’s career aspirations in STEM. Next, when female professors were seen as positive role models, women automatically identified with science and stereotyped science as more feminine than masculine. Moreover, viewing professors as positive role models was associated with pro-science career aspirations and attitudes (both implicit and explicit), for men and women alike. The findings suggest that female science professors benefit women provided students identify with them as role models. We conclude that female STEM professors not only provide positive role models for women, but they also help to reduce the implicit stereotype that science is masculine in the culture-at-large. We further discuss how shifting implicit gendered stereotypes about science can impact women’s investment in a science career.

Neurite Outgrowth and Growth Cone Morphology on Micropatterned Surfaces

The quantitative effects of micropatterned laminin surfaces on neurite outgrowth and growth cone morphology were investigated. Using microlithography, 20‐ or 30‐μm‐wide laminin stripes were applied to the surface of a glass coverslip, alternating with BSA‐coated glass stripes of the same dimension. Growth on these surfaces was strongly biased in the direction parallel to the stripes, but the mean length of outgrowth was reduced relative to that on uniform laminin surfaces. Growth cones were slightly more elongated on micropatterned surfaces than on controls and were aligned with the pattern. These results provide a starting point for examining the fundamental effects of micropatterned surfaces on neurite outgrowth and ways in which these may be useful in controlling and guiding neurite outgrowth for biotechnological applications.

Oriented Schwann cell monolayers for directed neurite outgrowth.

Schwann cells are an important component of the peripheral nervous system and participate in peripheral nerve regeneration. They create a supportive environment for neurite outgrowth by releasing trophic factors and up-regulating permissive molecules on their surface. In addition, Schwann cells are able to self-organize into linear arrays in vitro and in vivo, suggesting a possible role in neurite guidance. Previously, we showed that Schwann cell placement and orientation in subconfluent cultures can be controlled using microlithographically patterned laminin substrates (Thompson, D. M., and H. M. Buettner. Tissue Eng. 7(3):247–266, 2001). In the current study, these substrates were used to create oriented Schwann cell monolayers. Both Schwann cell orientation and coverage were quantified in response to seeding density, culture medium, and micropattern dimensions. In serum-free medium, increasing the seeding density yielded a linear increase in coverage of the substrate area but decreased cell alignment. In an alternate approach, Schwann cells were first seeded in serum-free medium at moderate seeding density, allowed to align, then expanded in serum-containing growth medium. This produced complete coverage without large seeding densities while preserving alignment to the micropattern. Alignment and coverage were unaffected by micropattern dimensions. This work provides a useful methodology for investigating Schwann cell guidance effects on growing neurites.

Stochastic dynamics of the nerve growth cone and its microtubules during neurite outgrowth

The controlled extension of neurites is essential not only for nervous system development, but also for effective nerve regeneration after injury. This process is critically dependent on microtubule assembly since axons fail to elongate in the presence of drugs which disrupt normal assembly dynamics. For this reason, neurite outgrowth is potentially controllable by manipulation of the assembly state of the intracellular array of microtubules. Therefore, understanding how microtubule assembly dynamics and neurite outgrowth are coupled, in the absence of drugs, can lend valuable insight into the control and guidance of the outgrowth process. In the present study we characterized the stochastic dynamics of neurite outgrowth and its corresponding microtubule array, which advances concomitantly with the advance of the nerve growth cone, the highly motile structure at the terminus of the growing neurite, using reported fluorescent microscopic image sequences (Tanaka and Kirschner, 1991, J. Cell Biol. 115:345–363). Although previously modeled as an uncorrelated random walk, the stochastic advance of the growth cone was found to be anticorrelated over a time scale of ∼4 min, meaning that growth cone advances tended to be followed by growth cone retractions ∼4 min later. The observed anticorrelation most likely reflects the periodic stops and starts of neurite outgrowth that have been reported anecdotally. A strikingly similar pattern of anticorrelation was also identified in the advance of the growth cones microtubule array. Cross‐correlation analysis showed that growth cone dynamics tended to precede microtubule dynamics on a time scale of ∼0–2 min, while microtubules tended to precede growth cone dynamics on a ∼0–20‐s time scale, indicating a close temporal coupling between microtubule and growth cone dynamics. Finally, the scaling of the mean‐squared displacements with time for both the growth cone and microtubules suggested a fractional Brownian motion model which accounts for the observed anticorrelation of growth cone and microtubule advance.

Measurement of leukocyte motility and chemotaxis parameters with the Millipore filter assay

Although in vitro assays have been widely used to study leukocyte chemotactic migration, finding the best way to quantitate these assays has proven to be an elusive goal. Investigators have usually resorted to reporting quantities such as the leading front distance, total migrating cells or number of cells past a given distance from their starting point. While these measures may often provide a valid comparison of cell migration under specific assay conditions, they also reflect physical characteristics of the assay that are irrelevant to the basic phenomenon of interest; thus, typical quantities measured in the assay are not useful for comparison between different systems or for correlation with in vivo performance. Recently, however, Tranquillo et al. (1988) demonstrated the utility of an analysis of the under-agarose assay in which the density profile of migrating cells is characterized in terms of two parameters: the random motility coefficient, mu, and the chemotaxis coefficient, chi. These parameters do reflect intrinsic cell movement independently of extraneous physical conditions. The analysis relies primarily on matching theoretical cell density profiles, calculated from a mathematical model in which mu and chi appear, to cell density profiles measured experimentally in the assay. In this paper, we extend the work of Tranquillo et al. to show that the same model can be applied successfully to the Millipore filter assay. In addition, we present measured values of mu and chi for rabbit polymorphonuclear leukocytes (PMNs) in response to, and as a function of the concentration of, the peptide attractant formyl-norleucyl-leucyl-phenylalanine (FNLLP). We also examine the relationship between results obtained for the filter assay and the under-agarose assay and consider the mechanistic basis of the parameters mu and chi.

Growth Cone Pathfinding: a competition between deterministic and stochastic events

BackgroundGrowth cone migratory patterns show evidence of both deterministic and stochastic search modes.ResultsWe quantitatively examine how these two different migration modes affect the growth cones pathfinding response, by simulating growth cone contact with a repulsive cue and measuring the resultant turn angle. We develop a dimensionless number, we call the determinism ratio Ψ, to define the ratio of deterministic to stochastic influences driving the growth cones migration in response to an external guidance cue. We find that the growth cone can exhibit three distinct types of turning behaviors depending on the magnitude of Ψ.ConclusionsWe conclude, within the context of these in silico studies, that only when deterministic and stochastic migration factors are in balance (i.e. Ψ ~ 1) can the growth cone respond constructively to guidance cues.