Brian A. Couch

N-Myristoylated c-Abl Tyrosine Kinase Localizes to the Endoplasmic Reticulum upon Binding to an Allosteric Inhibitor

Allosteric kinase inhibitors hold promise for revealing unique features of kinases that may not be apparent using conventional ATP-competitive inhibitors. Here we explore the activity of a previously reported allosteric inhibitor of BCR-Abl kinase, GNF-2, against two cellular isoforms of Abl tyrosine kinase: one that carries a myristate in the N terminus and the other that is deficient in N-myristoylation. Our results show that GNF-2 inhibits the kinase activity of non-myristoylated c-Abl more potently than that of myristoylated c-Abl by binding to the myristate-binding pocket in the C-lobe of the kinase domain. Unexpectedly, indirect immunofluorescence reveals a translocation of myristoylated c-Abl to the endoplasmic reticulum in GNF-2-treated cells, whereas GNF-2 has no detectable effect on the localization of non-myristoylated c-Abl. These results indicate that GNF-2 competes with the NH2-terminal myristate for binding to the c-Abl kinase myristate-binding pocket and that the exposed myristoyl group accounts for the localization to the endoplasmic reticulum. We also demonstrate that GNF-2 can inhibit enzymatic and cellular kinase activity of Arg, a kinase highly homologous to c-Abl, which is also likely to be regulated through intramolecular binding of an NH2-terminal myristate lipid. These results suggest that non-ATP-competitive inhibitors, such as GNF-2, can serve as chemical tools that can discriminate between c-Abl isoform-specific behaviors.

Synaptic Clustering of PSD-95 Is Regulated by c-Abl through Tyrosine Phosphorylation

The c-Abl tyrosine kinase is present in mouse brain synapses, but its precise synaptic function is unknown. We found that c-Abl levels in the rat hippocampus increase postnatally, with expression peaking at the first postnatal week. In 14 d in vitro hippocampal neuron cultures, c-Abl localizes primarily to the postsynaptic compartment, in which it colocalizes with the postsynaptic scaffold protein postsynaptic density protein-95 (PSD-95) in apposition to presynaptic markers. c-Abl associates with PSD-95, and chemical or genetic inhibition of c-Abl kinase activity reduces PSD-95 tyrosine phosphorylation, leading to reduced PSD-95 clustering and reduced synapses in treated neurons. c-Abl can phosphorylate PSD-95 on tyrosine 533, and mutation of this residue reduces the ability of PSD-95 to cluster at postsynaptic sites. Our results indicate that c-Abl regulates synapse formation by mediating tyrosine phosphorylation and clustering of PSD-95.

Increased Dendrite Branching in AβPP/PS1 Mice and Elongation of Dendrite Arbors by Fasudil Administration

Amyloid-beta (Abeta) overproduction and dendrite arbor atrophy are hallmarks of Alzheimers disease. The RhoA GTPase (Rho) signals through Rho kinase (ROCK) to control cytoskeletal dynamics and regulate neuron structure. Hyperactive Rho signaling destabilizes neurons leading to dendritic regression that can be rescued by genetic or pharmacological reduction of ROCK signaling. To understand what effect reduced ROCK signaling has on the dendrite arbors of mice that overproduce Abeta, we administered the ROCK inhibitor fasudil to AbetaPP/PS1 transgenic mice. We report that increased dendrite branching occurs in AbetaPP/PS1 mice and that fasudil promotes lengthening of the dendrite arbors of CA1 pyramidal neurons.

The Molecular Biology Capstone Assessment: A Concept Assessment for Upper-Division Molecular Biology Students

This article describes the development of the Molecular Biology Capstone Assessment, a multiple-true/false concept assessment targeted to upper-division students. This assessment is intended to help pinpoint areas of conceptual difficulty among graduating majors in order to facilitate curricular change at the departmental level.

Scientific Teaching: Defining a Taxonomy of Observable Practices.

The authors describe the development of a taxonomy detailing core goals and practices of Scientific Teaching (ST). This taxonomy will support future educational efforts by providing an empirical framework for researchers studying the processes and outcomes of ST-based course transformations as well as a concise guide for faculty developing classes.

The Abl and Arg non-receptor tyrosine kinases regulate different zones of stress fiber, focal adhesion, and contractile network localization in spreading fibroblasts.

Directed cell migration requires precise spatial control of F‐actin‐based leading edge protrusion, focal adhesion (FA) dynamics, and actomyosin contractility. In spreading fibroblasts, the Abl family kinases, Abl and Arg, primarily localize to the nucleus and cell periphery, respectively. Here we provide evidence that Abl and Arg exert different spatial regulation on cellular contractile and adhesive structures. Loss of Abl function reduces FA, F‐actin, and phosphorylated myosin light chain (pMLC) staining at the cell periphery, shifting the distribution of these elements more to the center of the cell than in wild‐type (WT) and arg−/− cells. Conversely, loss of Arg function shifts the distribution of these contractile and adhesion elements more to the cell periphery relative to WT and abl−/− cells. Abl/Arg‐dependent phosphorylation of p190RhoGAP (p190) promotes its binding to p120RasGAP (p120) to form a functional RhoA GTPase inhibitory complex, which attenuates RhoA activity and downstream pMLC and FA formation. p120 and p190 colocalize both in the central region and at the cell periphery in WT cells. This p120:p190 colocalization redistributes to a more peripheral distribution in abl−/− cells and to a more centralized distribution in arg−/− cells, and these altered distributions can be restored to WT patterns via re‐expression of Abl or Arg, respectively. Thus, the altered p120:p190 distribution in the mutant cells correlates inversely with the redistribution in adhesions, actin, and pMLC staining in these cells. Our studies suggest that Abl and Arg exert different spatial regulation on actomyosin contractility and focal adhesions within cells.

Student Buy-In to Active Learning in a College Science Course

Student buy-in as a key mechanism for student engagement and performance in an active-learning context is explored. This paper provides the first operational definition of student buy-in to in-class activities, in this case characterizing the complex nature of students’ responses in an active-learning classroom.

How Question Types Reveal Student Thinking: An Experimental Comparison of Multiple-True-False and Free-Response Formats

A comparison of questions posed in multiple true–false and free-response formats reveals how the question type affects diagnosis of the degree to which students hold correct and incorrect understandings.

Delayed amyloid plaque deposition and behavioral deficits in outcrossed AβPP/PS1 mice.

Alzheimers disease (AD) is a progressive neurodegenerative dementia characterized by amyloid plaque accumulation, synapse/dendrite loss, and cognitive impairment. Transgenic mice expressing mutant forms of amyloid‐β precursor protein (AβPP) and presenilin‐1 (PS1) recapitulate several aspects of this disease and provide a useful model system for studying elements of AD progression. AβPP/PS1 mice have been previously shown to exhibit behavioral deficits and amyloid plaque deposition between 4–9 months of age. We crossed AβPP/PS1 animals with mice of a mixed genetic background (C57BL/6 × 129/SvJ) and investigated the development of AD‐like features in the resulting outcrossed mice. The onset of memory‐based behavioral impairment is delayed considerably in outcrossed AβPP/PS1 mice relative to inbred mice on a C57BL/6 background. While inbred AβPP/PS1 mice develop deficits in radial‐arm water maze performance and novel object recognition as early as 8 months, outcrossed AβPP/PS1 mice do not display defects until 18 months. Within the forebrain, we find that inbred AβPP/PS1 mice have significantly higher amyloid plaque burden at 12 months than outcrossed AβPP/PS1 mice of the same age. Surprisingly, inbred AβPP/PS1 mice at 8 months have low plaque burden, suggesting that plaque burden alone cannot explain the accompanying behavioral deficits. Analysis of AβPP processing revealed that elevated levels of soluble Aβ correlate with the degree of behavioral impairment in both strains. Taken together, these findings suggest that animal behavior, amyloid plaque deposition, and AβPP processing are sensitive to genetic differences between mouse strains. J. Comp. Neurol., 521:1395–1408, 2013.

Characterizing Student Perceptions of and Buy-In toward Common Formative Assessment Techniques.

Formative assessments aim to promote learning by developing a cycle in which students demonstrate their understanding and receive feedback. This mixed-methods study sought to identify the degree to which students perceive various in-class and out-of-class activities as accomplishing key objectives of formative assessment.