TY - JOUR
T1 - Stable interaction between G-actin and neurofilament light subunit in dopaminergic neurons
AU - Hao, R.
AU - Macdonald, R. G.
AU - Ebadi, M.
AU - Schmit, J. C.
AU - Pfeiffer, R. F.
N1 - Funding Information:
Acknowledgements--The authors gratefully acknowledge the exceptional secretarial skills of Mrs Lori Clapper in preparing this manuscript. The authors extend their appreciation to Professor Robert B. Norgren, Jr of the Department of Anatomy and Cell Biology at the University of Nebraska College of Medicine in assisting with fluorescence microscopic analysis of dopaminergic neurons. The expert technical assistance of Dr Huaiyuen Han and Mr Jerry Lintz is gratefully acknowledged. This study was completed in part by a grant from United States Public Health Service NS 34566.
PY - 1997/12
Y1 - 1997/12
N2 - Excessive accumulation of neurofilaments in the cell bodies and proximal axons of motor neurons is a major pathological hallmark of motor neuron diseases. In this communication we provide evidence that the neurofilament light subunit (68 kDa) and G-actin are capable of forming a stable interaction. Cytochalasin B, a cytoskeleton disrupting agent that interrupts actin-based microfilaments, caused aggregation of neurofilaments in cultured mesencephalic dopaminergic neurons, suggesting a possible interaction between neurofilaments and actin; which was tested further by using crosslinking reaction and affinity chromatography techniques. In the cross-linking experiment, G-actin interacted with individual neurofilament subunits and covalently cross-linked with disuccinimidyl suberate, a homobifunctional cross-linking reagent. Furthermore, G-actin was extensively cross-linked to the light neurofilament subunit with this reagent. The other two neurofilament subunits showed no cross-linking to G-actin. Moreover, neurofilament subunits were retained on a G-actin coupled affinity column and were eluted from this column by increasing salt concentration. All three neurofilament subunits became bound to the G-actin affinity column. However, a portion of the 160 and 200 kDa neurofilament subunits did not bind to the column, and the remainder of these two subunits eluted prior to the 68 kDa subunit, suggesting that the light subunit exhibited the highest affinity for G-actin. Moreover, neurofilaments demonstrated little or no binding to F-actin coupled affinity columns. The phosphorylation of neurofilament proteins with protein kinase C reduced its cross-linking to G-actin. The results of these studies are interpreted to suggest that the interaction between neurofilaments and actin, regulaled by neurofilament phosphorylation, may play a role in maintaining the structure and hence the function of dopaminergic neurons in culture.
AB - Excessive accumulation of neurofilaments in the cell bodies and proximal axons of motor neurons is a major pathological hallmark of motor neuron diseases. In this communication we provide evidence that the neurofilament light subunit (68 kDa) and G-actin are capable of forming a stable interaction. Cytochalasin B, a cytoskeleton disrupting agent that interrupts actin-based microfilaments, caused aggregation of neurofilaments in cultured mesencephalic dopaminergic neurons, suggesting a possible interaction between neurofilaments and actin; which was tested further by using crosslinking reaction and affinity chromatography techniques. In the cross-linking experiment, G-actin interacted with individual neurofilament subunits and covalently cross-linked with disuccinimidyl suberate, a homobifunctional cross-linking reagent. Furthermore, G-actin was extensively cross-linked to the light neurofilament subunit with this reagent. The other two neurofilament subunits showed no cross-linking to G-actin. Moreover, neurofilament subunits were retained on a G-actin coupled affinity column and were eluted from this column by increasing salt concentration. All three neurofilament subunits became bound to the G-actin affinity column. However, a portion of the 160 and 200 kDa neurofilament subunits did not bind to the column, and the remainder of these two subunits eluted prior to the 68 kDa subunit, suggesting that the light subunit exhibited the highest affinity for G-actin. Moreover, neurofilaments demonstrated little or no binding to F-actin coupled affinity columns. The phosphorylation of neurofilament proteins with protein kinase C reduced its cross-linking to G-actin. The results of these studies are interpreted to suggest that the interaction between neurofilaments and actin, regulaled by neurofilament phosphorylation, may play a role in maintaining the structure and hence the function of dopaminergic neurons in culture.
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U2 - 10.1016/S0197-0186(97)00027-2
DO - 10.1016/S0197-0186(97)00027-2
M3 - Article
C2 - 9413844
AN - SCOPUS:0030862132
SN - 0197-0186
VL - 31
SP - 825
EP - 834
JO - Neurochemistry International
JF - Neurochemistry International
IS - 6
ER -