Title: A methodology for specific disruption of microtubule polymerization into dendritic spines
Legend: (A) Section of a representative dendritic arbor with one spine boxed. (B) Representative dendritic spine (yellow) rich with f-actin (red filaments) being invaded by a polymerizing MT (green). Other noninvading MTs are shown in the dendrite shaft. (C) Dendritic spine (yellow) of neuron transfected with LifeAct-scramble (blue and gray fusion protein) that is appropriately localizing to actin filaments (red) but does not affect the likelihood of a MT (green) directly polymerizing into the dendritic spine. (D) Dendritic spine (yellow) of neuron transfected with LifeAct-MTED (pink and gray fusion protein) that has localized to actin filaments (red) in the spine head and neck. LifeAct-MTED is shown depolymerizing a MT (green) before its entry into the dendritic spine. Created with BioRender.com. Lifeact- an actin-binding peptide, MTED – microtubule elimination domain peptide, mScarlet – a red fluorescent protein.
Citation: Holland, E. D., Miller, H. L., Millette, M. M., Taylor, R. J., Drucker, G. L., & Dent, E. W. (2024). A methodology for specific disruption of microtubule polymerization into dendritic spines. Molecular biology of the cell, 35(6), mr3. https://doi.org/10.1091/mbc.E24-02-0093
Abstract: 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.
Investigator: Erik W. Dent, PhD
About the Lab: The nervous system is a fantastically complex organization of neurons and non-neuronal cells that allows organisms to sense the environment, initiate movement and produce consciousness. We are interested in understanding how the nervous system develops and functions, specifically the central nervous system (CNS) (i.e. the brain and spinal cord). Two regions of the brain that we focus on are the cortex and hippocampus.