Current Research

The process of nuclear migration is essential to the growth, development, and function of all eukaryotes.  Essential developmental steps in Drosophila require the localization of nuclei to the periphery of the embryo and in the budding yeast, Saccharomyces cerevisiae,accurate nuclear positioning is necessary for correct segregation of genetic material.  Moreover, precise nuclear positioning is required for the asymmetrical cell division that occurs in early embryos of vertebrates.  It has been suggested that movement of the nucleus is a critical step for the migration of cancer cells and subsequent malignancy.

However, the actual mechanisms responsible for signaling the movement and regulating the positioning of nuclei within cells remain poorly understood.  The pioneering work of the laboaratory of Dr. Ron Morris at the Robert Wood Johnson Medical School of UMDNJ led to the identification of several genes involved in this process in the filamentous fungus Aspergillus nidulans [Ron has written a great review article too - see abstract].  A. nidulans is ideal for the analysis of nuclear migration because it is a multinucleated organism in which nuceli migrate, following division, from a central spore out into newly formed filaments during the life cycle of the fungus.  This enabled the isolation of several mutants which were defective in this process.  These genes were termed "nud" for defective in "nuclear distribution".  Whereas A. nidulans remains a most useful model for the study of these genes, it is limited as a single-celled organism and cannot tell us about the function of these genes in different cell-types or higher eukaryotes.  We are therefore utilizing the nematode Caenorhabditis elegans as a model organism to examine the expression and mechanism by which these gene act in a multicellular context.  This is most interesting from a medical standpoint because one of the genes found to be involved in A. nidulans nuclear distribution shares high DNA sequence identity with a human gene that has been implicated as the cause of a severe brain disorder called lissencephaly.  This childhood birth defect results in children with "smooth brains" that lack the stereotypical folds commonly associated with brain morphology.  These unfortunate individuals live a short lifetime filled with epileptic seizures and are severely mentally retarded.

Since fungi don't have nervous systems and nematodes do, we are using C. elegans to understand the possible action of this gene, and related genes, in neuronal development as well as other cellular processess.We have used the genetic information provided by the C. elegans Genome Consortium to facilitate the identification of worm homologs of A. nidulans nud genes.   Below is a composite photograph of an A. nidulans mutant strain, nudCts, that is defective in nuclear migration.  The panel on the top left shows a nudCmutant strain in which the nuclei (stained using DAPI and viewed under a fluorescent scope) are clumped in the spore, while on the top right panel (as viewed under phase-contrast) the filaments still have grown out fine.  In the bottom set of panels, the normal "wild-type" spacing of nuclei is restored to a  nudCts strain when it is transformed with a plasmid expressing the C. elegans homolog of nudC.  This experiment indicates that the nematode gene can function identically when transplanted in the fungus and suggests a common cross-species function for this gene among eukaryotes.

In order to better define where this gene might act in a multicellular organism such as C. elegans, we are using gene expression techniques that employ GFP, the naturally Green Fluorescent Protein from jellyfish.  A gene fusion between the regulatory elements controlling expression of the C. elegans nudC homolog (which we have named nud-1) and GFP indicates that this gene is highly expressed in neurons of the worm's head (pictured below at the right) and tail.  Therefore, this gene represents a link between nuclear migration and neuronal function.

We have also determined the expression pattern of the worm homolog of the human lissencephaly-causing gene (LIS-1).  In C. elegans, the lis-1 gene is highly expressed throughout the major neuronal processes of the animal. Interestingly, the non-neuronal expression of C. elegans lis-1 appears associated with cell-types that have more than one nucleus - reminiscent of Aspergillus nidulans.This may indicate a role for this gene in positioning of nuclei in a variety of species and cell-types. A paper that we have published on this evolutionary conservation of nuclear positioning gene function appeared in the evo-devo journal Development, Genes and Evolution. 

We are exploring the function of these gene products using recently developed technologies, such as double-stranded RNA-mediated inhibition (RNAi), the yeast two-hybrid system, and gene knockouts - in addition to classical genetics.  Our experiments indicate these genes participate in a variety of interesting cellular processes including proper embryo, gonad, and neuronal development.  We have also been able to induce lis-1 specific epileptic seizures in C. elegans (see Caldwell Lab Video Page) and are using this fascinating phenotype to delineate the possible causes of human epilepsy as it is associated with this lissencephaly or other neuronal disorders. This work was highlighted in an international press release from the Howard Hughes Medical Institute and was published in Human Molecular Genetics.  Our research into neurodevelopment is supported by a grant from the National Science Foundation's CAREER Award Program and involves graduate students and talented undegraduates who are attempting to identify additional genes linked to genetic causes of epilepsies and neuronal malfunction.  

To facilitate this work, we have developed a new comprehensive database for the genetics of epilepsy called CarpeDB.  This database is designed to serve the entire epilepsy and neurogenetics community.  CarpeDB is listed in the National Center for Biotechnology Information (NCBI) Molecular Biology Database Collection, published in Nucleic Acids Research and it was highlighted in Science magazine.  Check it out....and Seize The Data!

The photo on the left (courtesy of Tim Schedl's lab) is an example of one place in C. elegans where nuclei exist in "syncitium" or multinucleate mass.  This is a part of the worm's gonad that has been dissected out the animal and stained with a dye (DAPI) that allows for visualizaton of the DNA within individual nuclei.  Among the phenotypes we can observe when we disrupt the function of either the C. elegans lis-1 or nud-1 genes is sterility.  It is possible this phenotype is related to dysfunctional oogenesis, migration of nuclei, or nuclear positioning in related cell types within the gonads of genetically altered animals.  We are currently exploring all these options this in our laboratory and in collaboration with Dr. Jen Miskowski at the University of Wisconsin-La Crosse.

Another consequence of inhibiting nud-1 or lis-1function is embryonic lethality.  The video on the right shows a C. elegans embryo going through its  earliest cell divisions.   During this process the nuclei within the embryo are repositioned and divide in a characteristic pattern.  Proteins that function in proper postitioning and migration of nuclei may act in this mechanism of establishing embryonic polarity during cell division as well.   Click here to see our digital time-lapse videos showing the effects of depleting these genes on C. elegans embryonic cell division.  Understanding these basic defects may shed light on processes pertaining to aberrant cell division - a major consequence of which are cancers.  We are collaborating with Dr. Li Yu-Lee at the Baylor College of Medicine in Houston to examine similar features of cell division in mammalian cell cultures.  Further investigation into the roles played by such proteins, in a variety of cell-types, in addition to the identification of gene products with which they interact, will shed light on potentially novel aspects of nuclear dynamics and function in eukaryotes.  This work has  been published in The Journal of Cell Science.

This work has been generously supported by a National Science Foundation CAREER Award and a Basil O'Connor Scholar's Award from The March of Dimes Birth Defects Foundation.

Use of C. elegans to Investigate the Molecular Basis of Human Movement Disorders

Dystonia is a movement disorder consisting of involuntary twisting muscular contractions or abnormal postures.  Dystonia is estimated to be six times more prevalent than Huntington's Disease, ALS, or Muscular Dystrophy (NIH Budget Office), yet this severely incapacitating disease is often clinically misdiagnosed and remains poorly understood.  At least 14 different hereditary forms of dystonia have been identified, but a cure has not been found yet. A major breakthrough toward understanding the underlying cause of the most severe form of this disease,early-onset torsion dystonia, which affects all muscle groups in childhood, came with the identifcation of the gene linked to this disease, TOR1A (DYT-1), by Ozelius et al. in 1997.   This gene encodes a protein of unknown function called torsinA, which appears to be related to a family of proteins that are present in humans, rats, mice, and C. elegans.  The completion of the genomic DNA sequence for C. elegans has enabled us to identify three members of the torsin protein family that exhibit high sequence similarity to humans.  

We are utilizing all the molecular and genetic tools available to us in C. elegans to gain an understanding of this biologically significant family of proteins.  C. elegans,with its full genome sequence determined, a completely mapped neuronal connectivity diagram, and a plethora of genetic information represents an ideal model system for probing the functional relationships between these proteins and neuronal activity.   In this regard, we have already undertaken of torsin protien activity using a combination of RNA-interference (RNAi), genetic screens for interacting proteins, and biochemical approaches.  We are also using C. elegans to screen for small molecules that can cross the blood-brain barrier of humans and may influence torsin activity in cells.  Torsin proteins also share distant sequence similarity to molecular chaperones, molecules that assist proteins fold properly.  We have adopted an in vivo assay system to examine the ability of torsins to affect protein aggregation  - a common problem associated with neurological dysfunction, as it is associated with Alzheimer's, Huntington's,  Prion diseases and Parkinson's Disease.  Notably, torsins are localized to sites of protein aggregation called Lewy Bodies in the brains of people who have died from Parkinson's Disease.  Our combined genetic and genomic approach not only represents an attempt to further delineate the molecular basis of primary dystonia and Parkinson's Disease, but also to gain an insight into this novel class of evolutionarily conserved proteins found only in metazoans.  For more information please see our article, the cover story of February 2003 issue of Human Molecular Genetics.  

 

Other feature stories about our work into movement disorders can be found on the webpages of the National Institute of Neurological Disease and Stroke, the Howard Hughes Medical Institute, The Scientist, and Biophotonics International. This research has now been expanded to include an investigation of torsin activity in protecting dopamine neurons from cellular forms of stress linked to Parkinson's Disease (PD).   We are currently exploring several avenues toward understanding the molecular basis for Parkinson's including student-led screens for novel genetic and environmental factors that influence onset and progression of PD.   In collaboration with the laboratory of Dr. Susan Lindquist at The Whitehead Institute, we are investigating a previously unforseen cellular pathway that may be responsible for the early cellular defects that lead to Parkinson's Disease.  One of of the genes in this pathway, called Rab1, is naturally protective to cells that have been genetically engineered to mimic the cytotoxcity associated with PD.  This finding was corroborated across several species ranging from yeast, to fruit flies, worms, and rat neurons.   This work was reported in Science magazine and is discussed in this press release from the Howard Hughes Medical Institute.   Our collaborative work with Dr. Lindquist continues in this area and is fortified by other work to identify drugs that work within this mechanism in collaboration with the pharmaceutical industry.   This approach is also complemented by our involvement in the STOP-PD Program of The Parkinson's Institute.   This latter effort involves an exciting multi-institutional "pipeline" toward finding a small molecule that halts or reverses the progression of dopamine neuron loss by focusing on preventing the misfolding of human alpha-synuclein, a key protein that is linked to development of PD in patients.

For additonal information on dystonia or PD please see the very informative webpages of the Dystonia Medical Research Foundation, Parkinson's Disease Foundation, National Parkinson Foundation, The National Institutes of Health, The Bachmann-Strauss Dystonia and Parkinson Foundation and The Michael J. Fox Foundation - all of whom have generously funded this research in our laboratory.  A nice story about our grant from The Michael J. Fox Foundation can be found here.

Other Research Ongoing in The Shack...

We are always expanding the horizons of projects and interdiscipinary efforts here in The Worm Shack.....some of our current investigations include:

• Collaborative studies with researchers at The Mayo Clinic and UCLA into using worms to investigate factors influencing the degradation of proteins linked to Alzhiemer's Disease and dementia
  
• Work with the UAB Gene Therapy Center to examine worms as a vehicle to pre-test factors influencing success of human gene therapy targets
• An ongoing collaboration with The University of Alabama Department of Chemical and Biological Engineering to investigate use of magnetic nanoparticles for gene knockdown and delivery
• Molecular toxicology of ionic liquids, a class of important "green chemicals" that may serve to supplant organic solvents in manufacturing processes (in collaboration with the lab of Dr. Robin Rogers, UA Center for Green Manufacturing