Overexpression of neurofilament H disrupts normal cell structure and function.
Studying exogenously expressed tagged proteins in live cells has become a standard technique for evaluating protein distribution and function. Typically, expression levels of experimentally introduced proteins are not regulated, and high levels are often preferred to facilitate detection. However, overexpression of many proteins leads to mislocalization and pathologies. Therefore, for normative studies, moderate levels of expression may be more suitable. To understand better the dynamics of intermediate filament formation, transport, and stability in a healthy, living cell, we inserted neurofilament heavy chain (NFH)-green fluorescent protein (GFP) fusion constructs in adenoviral vectors with tetracycline (tet)-regulated promoters. This system allows for turning on or off the synthesis of NFH-GFP at a selected time, for a defined period, in a dose-dependent manner. We used this inducible system for live cell imaging of changes in filament structure and cell shape, motility, and transport associated with increasing NFH-GFP expression. Cells with low to intermediate levels of NFH-GFP were structurally and functionally similar to neighboring, nonexpressing cells. In contrast, overexpression led to pathological alterations in both filament organization and cell function.
Szebenyi
Györgyi
G
Department of Cell Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9039, USA.
Smith
George M
GM
Li
Ping
P
Brady
Scott T
ST
D016475:3T3 Cells; D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002468:Cell Physiological Phenomena / Q000187:drug effects*; D002477:Cells / Q000187:drug effects* / Q000648:ultrastructure*; D004305:Dose-Response Relationship, Drug; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D016900:Neurofilament Proteins / Q000235:genetics / Q000494:pharmacology*
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D016900:Neurofilament Proteins; C051774:neurofilament protein H; D049452:Green Fluorescent Proteins