Nuclear Protein Membrane Essay, Research Paper
1. The nuclear envelope is a double membrane extension of the rough endoplasmic reticulum containing many nuclear pore complexes. The lipid bilayer of the inner nuclear membrane is supported by the nuclear lamina,a mesh work of lamin filament located adjacent to the inside face of the nuclear envelope.(Lodish et al 1997) The lamina is proposed to be involved in both organizing chromatin and in DNA replication in interphase cells (Gerace and Burke, 1988). Localization and isolation of nuclear lamina done by Nancy Dwyer and Gunter Blobel that nuclear lamina is indeed associated to fractional equivalent of a peripheral layer beneath the inner membrane of the nuclear envelope. Lamin was not attributed to residual DNAase resistant heterochromatin, there was no DNA nor histone proteins observed in chromatographic assays of the membrane-nuclear-pore-lamin layers.(Blobel and Dwyer, 1976). In addition, utilizing electron microscopy, membrane portions cut perpendicular to the nuclear membrane surface have shown that the nuclear lamina layer is submembranous as opposed to intramembranous. Nuclear lamin is a type of intermediate filament protein which are divided into two major subgroups. One subgroup contains NL-A and NL-C which are soluble’ during cell division, whereas the other contains NL-B proteins which remain associated with lipid vesicles during mitosis( Nigg 1992 as cited in Goldman et al 1992). How can one devise/design and experiment which determines the intracellular route(pathway) by which nuclear laminin is targeted to the inner face of the nuclear membrane? Nuclear lamin like all nuclear proteins are constructs including sequences encoding for a nuclear localization signal sequence(NLS). First in order to determine whether or not lamins are indeed localized in the nucleus, it can be ascertained by modifying or deleting the NLS to insure whether or not lamin assembly in the nucleas was obtained. This can be obtained by cloning lamin B from whole ovary mRNA by RT-PCR. Afterwards creating mutants using PCR, and then sub-cloning the relevant sequences into pGEX-4T-3.( Ellis et al). These mutants are immunolabelled using fluorescein or maybe gold-conjugated anti-lamin antibodies. The lamins are then added to egg extracts prior to sperm pronuclear assembly. Observations should show that these mutated lamins fail to polymerize within the nuclear membrane. These experiments are conducted with a control lamin which should support nuclear lamina assembly. Lamin incorporation during the interphase stage of the cell cycle can also be observed. Recent experiments have traced the pathway of incorporation of microinjected lamin A into the Nuclear Envelope. By obtaining human NL-A through the expression of the appropriate cDNA and then immunolabelling them through biotinylation followed by microinjecting the lamins in the cytosol of 3T3 cells, antibiotin and then antilamin antibodies conjugated with flourescein were used to detect where staining occurred. These cells were contrasted with cells not microinjected and just labelled with flourescein conjugated anti-lamins to show that the microinjected lamins are indeed incorporated into the same area with the same ability to integrate within the nuclear lamina network. Observations also show that the nuclear lamin network in mouse 3T3 cells are capable of incorporating new NL into its constituent polymeric structure during interphase (Goldman et al 1992).
Following the above experiments, it can be observed that lamins are targeted to the nucleus and the inner membrane layer of the nuclear envelope. How can it be shown that the targetted lamins are indeed incorporated to the inner face of the inner layer of the nucleus? An experiment using a cell-free nuclear assembly extract derived from Xenopus eggs show that three components are needed for successful nuclear envelope formation. These three components consist of membranes, nuclear pores and a nuclear lamina. By taking fractionated nuclear extracts and adding them to a soluble solution containing sperm chromatin, metaphase chromosome, or DNA, intact nuclear envelopes begin to form. Addition of lamins to nuclear membrane components without chromatin do not initiate nuclear envelope formation, neither does lamin addition to chromatin alone. By depleting the lamin content in a cell free system does not prevent nuclear envelope formation but severely handicaps both its size and ability to replicate DNA. This was observed by monitoring the rate of incorporation of radiolabeled dCTP into DNA assembled int o nuclei. By blocking nuclear protein transport using lectin WGA it is shown through immunoflourescence that a protein lamin layer is not formed within the nucleus although nuclear envelope formation(including nuclear pores) is not blocked. By counteracting the effect of WGA, lamin polymerization in the nucleus can is then restored. The results show that lamin incorporation may not occur until an intact, actively transporting nuclear envelope has formed around chromatin(Newport et al 1990). The results show that a receptor, like a lamin B receptor proposed to be located in the inner nuclear membrane and an intact nuclear envelope capable of selective nuclear transport surrounding chromatin are needed for lamin assembly showing that lamin are targetted to the nucleus and specifically to the inner face of the nuclear membrane.1. Blobel, G., Dwyer, N., A Modified procedure for the isolation of a pore complex-lamina fraction from Rat Liver nuclei. J. Cell Biology. Vol 70, 1976 581-591.2. Burke, B., Gerace, L., Cell free system to study reassembly of the nuclear envelope at the end of mitosis. Cell Vol 44 February 28, 1986 639-652.3. Ellis, D.J., Jenkins, H., Hutchison, C.J., Whitfield, W.G., GST-lamin fusion proteins act as dominant negative mutants in Xenopus egg extract and reveal the function of the lamina in DNA replication. J. Cell Science. Vol 110 October 7,1997, 2507-2518.4. Goldman, R.D., Goldman, J.E., Moir, R.D., Montag-Lowy, M., Stewart, M. Pathway of Incorporation of Microinjected Lamin A into the Nuclear Envelope. J.Cell Biology, Vol 119, Number 4, November 1992, 725-734.5. Lodish, H, Baltimore, D, Berk, A, Matsudaira, P, Darnell, J, Zipursky, L., Molecular Cell Biology, New York: Scientific American Books, 1997.6. Newport, J.W., Dunphy, W.,G., Wilson, K.L. A Lamin-independent Pathway for Nuclear Envelope Assembly., J. Cell Biology, Vol 111, 2247-2259