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Revision as of 14:21, 8 May 2013
Lab 1 Activities
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The Origin of Eukaryotes from Prokaryotes by Time and Genetic Distance
Nick Lane Energetics and genetics across the prokaryote-eukaryote divide. Biol. Direct: 2011, 6();35 PMID:21714941
Copyright ©2011 Lane; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Endosymbiotic origin of eukaryotes by time and genetic distance 
Research Article Using Confocal Microscopy
The aim of this study was to investigate and provide evidence to support the migration and morphological change of mesothelial cells during tissue injury response. By using confocal microscopy imaging Nagai and peers, 2013, were able to observe clearly delineated mesothelial cells within ex vivo mouse abdominal wall tissue culture, as well as transformed mesothelial cells (MeT5A) in in-vivo tissue culture and witness their morphological transition from flat cells to cuboidal cells. This transition was able to be observed as confocal microscope analysis allows for the control in the depth of field and eliminates/reduces background matter, so that mesothelial cells positioned on top of each other can be differentiated. In addition, these confocal microscopy techniques also allowed the researchers to observe the horizontal migration of these mesothelial cells across the tissue culture, moving through gaps between surrounding cells. These findings provided evidence in support of the change and migration of mesothelial cells during injury response. 
--Mark Hill (talk) 12:05, 11 April 2013 (EST) This is good explanation as to how the confocal microscope was used in this research paper, the Pubmed referencing format is correct, you should have inserted at the beginning of paragraph (after study) rather than at the end. Your link at the top does not work.
Nuclear envelope breakdown in starfish oocytes proceeds by partial NPC disassembly followed by a rapidly spreading fenestration of nuclear membranes 
Lenart and co-workers (2003)examined the breakdown of the nuclear envelope in live starfish oocytes using various techniques involving fluorescently labeled dextrans (polysaccharides), membrane dyes and green fluorescently tagged proteins (GFP-tagged proteins), in conjunction with confocal time-lapse microscopy and electron microscopy which allowed them to view the changes in the oocyte nuclear envelope. Through the use of different sized fluorescently labelled dextran fractions experimenters were able to analyse the changes in the permeability of the nuclear envelope in maturing starfish oocytes. They firstly observed a sequential entry of dextran molecules, beginning with the smaller molecules entering the nucleus followed by larger molecules with diameters up to 40nm. The use of GFP-tagged nucleoporins revealed a corresponding gradual loss of peripheral nucleoporins from Nuclear Pore Complexes (NPC) (selective aqueous channels that span both nuclear membranes, connecting the nucleus with the cell cytoplasm) which resulted in the dilation of these channels from 10 to 40nm and a gradual release of import substrates. It was clearly noted, however, that the core of NPCs remained intact. Another key observation which was made was that the nuclear envelope structure remained unaffected. Next a rapid complete permeabilisation of the nuclear envelope that spread throughout the nuclear surface was noted, allowing macromolecules of diameters up to 100nm to enter the nucleus, this proposed the complete removal of the remaining core of the NPCs. It was also found that at the electron microscope level the nuclear envelope was fenestrated in the end. These experimenters therefore concluded that the breakdown of the nuclear envelope occurred in two distinct sequential phases:
-Phase 1 involves the gradual disassembly of the NPCs, which coincides with an increase in nuclear membrane/envelope permeability allowing for some macromolecules to enter the nucleus and nuclear import substrates to leave. At the end of this first phase the nuclear envelope remains intact.
-Phase 2 is characterised by an increase and rapid spreading nuclear envelope permeability as well as apparent fenestration of the nuclear envelope and hence its breakdown resulting from the removal of the core of NPCs. 
This research paper is relevant to the nuclear envelope breakdown section as it provides an experiment which clearly shows evidence of how the nuclear envelope gradually breaks down and explores the role of NPCs in this.
This paper explores the importance of the Nuclear Pore Complex (NPC) transmembrane nucleoporin, gp210, in the breakdown of the nuclear envelope. Galy and his colleagues subjected Caenorhabditis elegans (worm) egg extracts to RNAi-mediated deletion or mutation of the gp210 nucleoporin which resulted in the prevention of lamin depolymerisation, a late event which aids in the breakdown of the nuclear envelope. The prevention of lamin depolarisation results in the inability of the nuclear envelope to breakdown and also blocks pronulclear chromosome mixing that ultimately leads to the formation of two nuclei after mitosis. This implied that gp210 was key in nuclear envelope breakdown.
The block of gp210 phosphorylation which is conducted by cyclin-B-cdc2 (proposed to be an important trigger for the initiation of mitosis) was also explored. Depletion of the cyclin-B-cdc2, achieved through RNAi, resulted in the inability of the gp210 nucleoporin to be phosphorylated and achieve nuclear envelope breakdown. Hence, this provided further evidence that gp210, particularly its phosphorylation, was indeed an early step in the breakdown of the nuclear envelope.
This research article is relevant to the subheading Nuclear Envelope Breakdown as it suggests one of the nucleoporins involved in the breakdown of the nuclear envelope. 
In this study, Makhnevych et.al (2003) revealed a new mechanism for regulating transport into the nuclear compartment during closed mitosis in yeast. This mechanism involved specific molecular rearrangements in the nuclear pore complex (NPC). Karyopherins (kaps) are soluble transport factors that facilitate the transport of distinct cargo molecules into the nucleus by binding to NPCs. This transport can be regulated so as to conduct changes in gene transcription, DNA replication and chromosome segregation. Previous studies identified posttranslational modifications to the cargo molecules (including phosphorylation and acetylation), resulting in an increased or decreased affinity to their kap transporters, as a way of inhibiting or enhancing their transport into the nucleus. A mechanism which is less commonly examined as a form of import regulation of cargo molecules is the alteration of the kaps transport machinery and one of these mechanisms is explored in this paper. Makhnevych and peers specifically analysed the control of Kap121p related cargo molecules into the nucleus. They observed that Kap121p –mediated import is active in interphase but inhibited during mitosis and this was not due to changes in cargo binding to Kap121p. They found that by structurally rearranging the NPC during mitosis, a masked binding site for Kap121p would be revealed on one of its nucleoporins, Nup53p. When Kap121p was bound to this site, movement of Kap121p and its cargo into the nucleus was slowed and it triggered the release of the cargo into the cytoplasm. Hence, Nup53p was considered a transport inhibitory nup (iNUP). They concluded that the regulation of Kap121p-mediated import through the presence of Nup 53p played an important role in the progression through mitosis, showing that increased levels of Nup53p inhibited progression through mitosis.
This research paper is relevant to the subheading “Open vs Closed/semi-closed Mitosis” as it provides a model of closed mitosis and examines a mechanism used to regulate transport of molecules into the nucleus during mitosis which therefore shows the continuous presence of NPCs and hence highlighting the fact that the nuclear envelope stays intact during closed mitosis in lower eukaryotes such as yeast.
This article investigates the translocation of pre-existing nuclear pore complexes (NPC) from mother yeast cell to its bud during cell division and how this contributes to the aging of yeast cells. Shcheprova and peers (2008) used fluorescent tags and photobleaching to track the movement of cellular components including nucleoporins within the nuclear envelope. They observed that NPCs moved freely throughout the nuclear envelope, however, when part of the nucleus penetrated into the bud, NPC movement was restricted to the part of the nuclear envelope within the mother cell as a diffusion barrier was created at the neck of the bud. They established that NPCs are prevented from moving from the mother to the bud during cell division. They also observed that no new NPCs were inserted in the nuclear envelope of the mother and that the pre-existing NPCs remained in the mother, but there was evidence of new NPC (not from the mother) insertion in the bud. They concluded that the division of the yeast nucleus is asymmetric when considering the age of the pores with pre-existing material being preferentially segregated to the mother. 
This article is relevant to the “Open vs. Closed Mitosis” section as it addresses the fate of NPC during closed mitosis and highlights the fact they are not broken up like in open mitosis.
Antibody against Gap Junction Protein Alpha 1 (Connexin 43)
Type of Immunoglobulin: Polyclonal
Species it's raised in: Rabbit
Species it reacts against Human, Mouse and Rat
Application: Western Blot, Immunohistochemistry, Immunofluorescence and ELISA
Reference of use: Staphylococcus aureus-derived peptidoglycan induces Cx43 expression and functional gap junction intercellular communication in microglia
1) Do you see any change in phenotypes between group A and group B?
Yes. The majority of the control group WT undifferentiated B35 cells were of the broken fan and stumped phenotype, with very short lamellum/lamella and neutrite(s). On the other hand the majority of the Tm4 over-expressed undifferentiated B35 cells were of the pronged and stringed phenotype with multiple and longer neutrites and a single lamellum.
2) If you see a difference, speculate about a potential molecular mechanism that has lead to the change
It has been observed that Tm4 is found concentrated in the growth cones of neurons and in growing neutrites being involved in stabilizing the actin filaments and controlling the interactions between actin and actin binding proteins required for the expansion and motility of the growth cone,  hence, the over expression of this tropomyosin isoform would lead to increased actin-actin binding protein interactions that would enhance the motile events needed for neutrite growth. This would account for the increase in neutrite number and length in the Tm4 over-expressed cell group, giving the observed shift from a more broken fan/stumped phenotype to a more pronged/stringed phenotype in B35 cells