Molecular and Cell Biology Laboratory
Jesse and Caryl Philips Foundation Chair
Developmental and pathological changes in the organization and functions of the cell nucleus
The discovery of extremely long-lived proteins and their role in aging. As proteins age, they are more likely to encounter molecular damage. To combat the functional decline of the proteome, cells use the process of protein turnover by which potentially impaired polypeptides are constantly replaced with new functional copies. Consequently, a protein with a slow or no rate of turnover is at great risk to accumulate damage over extended periods of time. We have recently discovered extremely long-lived proteins (ELLPs) in post-mitotic cells of the rat central nervous system. Strikingly, these ELLPs are associated with chromatin, the nuclear envelope and the plasma membrane, all of which are cellular compartments that coordinate a myriad of regulatory functions. These proteins are therefore fully exposed to potentially harmful metabolites. Thus, we hypothesize that the failure to maintain proper levels and functional integrity of ELLPs in non-proliferative cells could be a major contributor to age-related changes in cell and tissue function. Our goal is to identify new ELLPs in various tissues of adult rats, study the biochemical properties of ELLPs in tissues of young and old animals, and test if there is a functional link between the decline of ELLPs and cellular aging. If successful our studies hold the promise of revealing new principles of protein homeostasis and age-related loss of cell function, both during "normal" aging and in age-related disease.
Age-related defects of nuclear transport channels: Changes in gene activity are part of the cellular aging process, however, the mechanisms that cause age-related alterations in gene expression are poorly understood. We have recently discovered that NPCs, essential multiprotein channels that mediate molecular trafficking between the nucleoplasm and cytoplasm of eukaryotic cells, are extremely long-lived in post-mitotic tissue and deteriorate over time causing a loss of cell compartmentalization in post-mitotic neurons. Our results suggest that nuclear pore deterioration might be a general aging mechanism leading to age-related defects in nuclear function, such as the loss of youthful gene expression programs. Age-dependent deterioration of nuclear pore complex function and the associated failure of the nuclear permeability barrier is characterized by the leaking of cytoplasmic proteins into the nucleoplasm. We detected large filaments inside the 'leaky' nuclei of old mouse and rat neurons, which stained with the cytoplasmic protein tubulin. Strikingly, tubulin-positive intranuclear structures have been linked to various neurological disorders including Parkinson's disease. Thus, nuclear pore deterioration might initiate or contribute to the onset of certain neurodegenerative diseases.
Loss of nuclear membrane integrity in human cancer cells. The nucleus houses most of the cell's genetic material and forms a defined compartment in which genomic functions can operate under the protective shield of the NE. Recently, we discovered a remarkable phenomenon whereby the nuclear envelope (NE) becomes transiently ruptured and repaired during interphase in various human cancer cells. This partial breakdown nuclear barrier is associated with the temporary loss of nuclear integrity and the entrapment of cytoplasmic organelles in the nucleus. The NE is a double membrane that regulates all nuclear trafficking of RNAs and proteins and prevents the passive diffusion of molecules larger than ~40 kDa between the nucleoplasm and the cytoplasm. Using fluorescent protein reporters in conjunction with time-lapse microscopy to monitor the integrity of the nuclear compartment in various cancer cell lines, we observed bursts of NE rupturing events, each followed by NE repair, during interphase in a subset of cells. Not only did cells survive these crises, they continued to proliferate. Strikingly, NE rupturing was associated with the formation of micronuclei, the mislocalization of nucleoplasmic proteins and, in the most extreme cases, the entrapment of mitochondria in the nuclear interior. The frequency of these NE rupturing events was higher in cells in which the nuclear lamina, a network of intermediate filaments providing mechanical support to the NE, was not properly formed. Our data uncover the existence of a NE instability that might induce random mutations including chromosome rearrangements and thereby promote tumor formation.
In summary, NE rupturing might dramatically change the genomic landscape of cancer cells and thus accelerate the rate at which evolving premalignant cells can acquire favorable genotypes instrumental for tumor progression. As such, NE instability might be a cancer-enabling feature relevant for cancer diagnostics and intervention.
The role of the nuclear pore complex in chromatin organization and gene regulation. Recently we established a new research area in the lab that focuses on the potential role of NPC components in gene regulation. Nuclear chromatin organization plays an important but poorly understood role in the establishment and maintenance of specific gene expression programs in eukaryotic cells. Using Drosophila melanogaster as a model system we discovered an unexpected link between the dynamic organization of NPCs and gene regulation. We have detected several mobile nucleoporins at sites of on-going transcription during development. Strikingly, these chromatin-bound nucleoporins can be found inside the nucleus. Furthermore, the recruitment of nucleoporins to active sites coincides with the onset of transcription and does not depend on the presence of mRNA. Significantly, depletion of these nucleoporins by RNAi resulted in a block of transcription of nucleoporin target genes. In addition, we found a genetic interaction between several nucleoporins and the formation of silent chromatin. Chromatin binding at specific loci, not associated with gene activity, was also observed for several other dynamic nucleoporins. Based on these findings we hypothesize that mobile nucleoporins are key players in gene regulation via their association with chromatin inside the nucleus.
In summary, our results may establish a novel link between nuclear membrane formation and chromatin organization. This could provide new insights into the well-known phenomenon of pathological nuclear architecture and the physiological consequences of aberrant nucleoporin expression observed in various cancer cells. Ultimately, our studies will lead to a better understanding of developmental gene expression and we hope to translate this knowledge into novel strategies to detect nuclei exhibiting aberrant gene activity and to control expression of disease-associated genes.
Ordinarily, the proteins known as nucleoporins are among the 30 gene products serving as the bricks and mortar of nuclear pore complexes, the communication channels that regulate the passage of molecules to and from a cell's nucleus. But recent research in Hetzer's laboratory suggests that some also have a second job of regulating specific genes during development, and that they sometimes stray from the straight and narrow to play a role in cancer as well.
For more than a decade, scientists have known that when the nucleoporin NUP98, which should be turned off during cell differentiation, abnormally fuses with certain proteins that regulate gene expression, the marriage causes leukemia. Moreover, in many cancers, the nucleoporins NUP214 and NUP88 are misregulated and in particular are associated with very aggressive forms of lung cancer.
Investigators have long questioned why these components of the cell's transport channel are implicated in cancer and have theorized that the connection relates to a problem in the conveyance of molecules in and out of the nucleus. But Hetzer offers a different explanation. He and his team believe these proteins also function as a new class of gene transcription regulators, which turn specifi c genes on and off during cell differentiation or tissue development. They found that nuclear pore proteins are not only part of the transport channels but also play a role in the organization of the genome and a very direct role in gene expression.
In another line of research, the Hetzer laboratory used metabolic pulse-chase labeling of whole animals in combination with quantitative mass spectrometry to identify and characterize extremely long-lived proteins (ELLPs) that reside in the nucleus. These proteins exhibit little to no protein turnover in the adult rat brain and thus escape the central mechanism that allows cells to maintain a functional proteome. This raises important questions about the biochemical properties of these proteins and whether the failure to maintain proper levels and functional integrity of ELLPs could be a major contributor to age-related changes in the function of post-mitotic tissues such as those of the brain and heart.