Biology subjects

Dent, E. W.

Publications and source records attributed to Dent, E. W..

4 recordsLinked to original sources

bioRxiv2024

F-BAR proteins CIP4 and FBP17 function in cortical neuron radial migration and process outgrowth

Neurite initiation from newly born neurons is a critical step in neuronal differentiation and migration. Neuronal migration in the developing cortex is accompanied by dynamic extension and retraction of neurites as neurons progress through bipolar and multipolar states. However, there is a relative lack of understanding regarding how the dynamic extension and retraction of neurites is regulated during neuronal migration. In recent work we have shown that CIP4, a member of the F-BAR family of membrane bending proteins, inhibits cortical neurite formation in culture, while family member FBP17 induces premature neurite outgrowth. These results beg the question of how CIP4 and FBP17 function in radial neuron migration and differentiation in vivo, including the timing and manner of neurite extension and retraction. Indeed, the regulation of neurite outgrowth is essential for the transitions between bipolar and multipolar states during radial migration. To examine the effects of modulating expression of CIP4 and FBP17 in vivo, we used in utero electroporation, in combination with our published Double UP technique, to compare knockdown or overexpression cells with control cells within the same mouse tissue of either sex. We show that either knockdown or overexpression of CIP4 and FBP17 results in the marked disruption of radial neuron migration by modulating neuronal morphology and neurite outgrowth, consistent with our findings in culture. Our results demonstrate that the F-BAR proteins CIP4 and FBP17 are essential for proper radial migration in the developing cortex and thus play a key role in cortical development. SIGNIFICANCE STATEMENTDuring embryonic development, radial migration of newly born cortical neurons is a complex process that underlies the proper formation of the neocortex, the outermost layers of neurons in the brain. Disruptions in radial migration results in profound effects on cognitive function and can lead to devastating developmental disabilities. To better understand this critical process in brain development we examined two members of the F-BAR family of membrane bending proteins, CIP4 and FBP17, which are present in the developing brain. We demonstrate that intracellular concentrations of these proteins must be tightly regulated. Increasing or decreasing levels of either protein has profound effects on neuronal morphology and proper radial migration, suggesting they are key players in cortical development.

neuroscience↗

bioRxiv2024

A Methodology for Specific Disruption of Microtubules in Dendritic Spines

Dendritic spines, the mushroom-shaped extensions along dendritic shafts of excitatory neurons, are critical for synaptic function and are one of the first neuronal structures disrupted in neurodevelopmental and neurodegenerative diseases. Microtubule (MT) polymerization into dendritic spines is an activity-dependent process capable of affecting spine shape and function. Studies have shown that MT polymerization into spines occurs specifically in spines undergoing plastic changes. However, discerning the function of MT invasion of dendritic spines requires the specific inhibition of MT polymerization into spines, while leaving MT dynamics in the dendritic shaft, synaptically connected axons and associated glial cells intact. This is not possible with the unrestricted, bath application of pharmacological compounds. To specifically disrupt MT entry into spines we coupled a MT elimination domain (MTED) from the Efa6 protein to the actin filament-binding peptide LifeAct. LifeAct was chosen because actin filaments are highly concentrated in spines and are necessary for MT invasions. Temporally controlled expression of this LifeAct-MTED construct inhibits MT entry into dendritic spines, while preserving typical MT dynamics in the dendrite shaft. Expression of this construct will allow for the determination of the function of MT invasion of spines and more broadly, to discern how MT-actin interactions affect cellular processes. Significance StatementO_LIThe LifeAct-MTED construct provides spatial and temporal control of microtubule depolymerization within individual cells C_LIO_LITargeting this construct directly to spines allows for the specific inhibition of microtubule dynamics into dendritic spines, without affecting microtubule dynamics in the dendritic shaft C_LIO_LIImplementation of the construct will allow for the testing of microtubule contributions to specific biological processes, without global disruption of microtubule dynamics C_LI

neuroscience↗

bioRxiv2023

Handling difficult cryo-ET samples: A study with primary neurons from Drosophila melanogaster

Cellular neurobiology has benefited from recent advances in the field of cryo-electron tomography (cryo-ET). Numerous structural and ultrastructural insights have been obtained from plunge-frozen primary neurons cultured on electron microscopy grids. With most primary neurons been derived from rodent sources, we sought to expand the breadth of sample availability by using primary neurons derived from 3rd instar Drosophila melanogaster larval brains. Ultrastructural abnormalities were encountered while establishing this model system for cryo-ET, which were exemplified by excessive membrane blebbing and cellular fragmentation. To optimize neuronal samples, we integrated substrate selection, micropatterning, montage data collection, and chemical fixation. Efforts to address difficulties in establishing Drosophila neurons for future cryo-ET studies in cellular neurobiology also provided insights that future practitioners can use when attempting to establish other cell-based model systems.

biophysics↗

bioRxiv2019

Double UP: efficient in utero electroporation via internal controls

In Utero Electroporation is a powerful tool for testing the role of genes in neuronal migration, but this technique suffers from high degrees of variability due to multiple factors. Therefore, we developed Double UP, a novel approach that combines LoxP-flanked reporters and limiting Cre dosages to generate internal controls. This technique allows for more rigorous quantification of migration, while decreasing the number of animals, reagents and time to complete experiments.

neuroscience↗