Mutations, Metabolism, Viruses & IF organization

In a study carried out with the help of then undergraduate Dorothy Plummer, we examined IF organization in fibroblasts derived from people with the genetic disease Giant Axonal Neuropathy (GAN).

This is an genetic disease, inherited in an autosomal dominant manner, which initially reveals as muscle weakness.

It is characterized by the presence of large accumulations of neuronal IFs in the distal parts of motoneurons.

That GAN was a defect in IF organization, rather than IFs themselves, was suggested by the observation that IF organization in a number of different cell types, expressing different IF proteins, was disrupted.

In particular, fibroblasts from GAN-patients displayed 'collapsed' vimentin filament networks.

We studied cell lines from such individuals and found, surprisingly, that vimentin organization was "conditional", that is it could be rescued by rather simple changes in culture conditions.

This result suggested that the GAN phenotype is due to a defect in metabolism rather than a defect in cytoskeletal systems.

Klymkowsky, M.W. & D.J. Plummer. 1985. Giant axonal neuropathy: A conditional mutation affecting cytoskeletal organization. J. Cell Biol.100: 245-250.

Klymkowsky, M.W., E. Christian, R.G. Ham, D.J. Plummer & F. Sprenger. 1988. Giant axonal neuropathy, intermediate filaments and cellular metabolism. in Intrinsic Determinants of Neuronal Form. Alan R. Liss, Inc., pp. 441-459.

It lead into an analysis of various metabolic affectors.

Reports at the time suggested that acrylamide could be used to specifically disrupt IF organization. 

In contrast, I found that acrylamide was an inhibitor of protein synthesis, and that other protein synthesis inhibitors also disrupted IF organization in cultured fibroblasts.

← roll over. 

A curious observation to emerge from these studies (and truth to tell, I love curious observations) was that treating fibroblasts with the protein kinase inhibitor H8 lead to a rapid change in morphology, from fibroblastic to neuronal.  

Frog virus 3 & IF organization

The absence of an overt phenotype following the disruption of IF organization in cultured cells led us to examine more arcane systems.

One of these was the frog virus 3 system.

Gopal Murti and his co-workers (at St. Jude's Research Hospital, Memphis) had previously shown that during FV3 infection, the cell's vimentin system "collapsed" and encircled the cytoplasmic viral assembly factories.

We found that disrupting IF organization, either by treatment of cells with anti-microtubule drugs, or more directly through anti-IF antibody injection, reduced the efficiency of viral assembly.

The process of IF encirclement bears superficial similarity to the association of vimentin filaments with lipid vesicles during adipocyte differentiation; that said, the effects of the absence of vimentin (in vimentin null mice) on fat cell physiology have not been reported.

Membranes & poliovirus replication

In a second viral-IF project was carried out in collaboration with Karla Kirkegaard (now at Stanford University).

During poliovirus infection, the infected cell's cytoskeleton is dramatically reorganized.

We asked whether the presence of IFs altered poliovirus replication, using the IF-free variant of the SW13 cell line developed and characterized by Bob Evans.

While the presence or absence of IFs had no apparent effect on viral repllication, we did in the course of these studies identify brefeldin A as a potent inhibitor of poliovirus replication.