UNSW Cell Biology

ANAT3231 Laboratory 06

Introduction

This laboratory is an opportunity to use online Medline and other Journal resources to study currrent research topics in the Cell Cytoskeleton.

Objectives

Understand how to access online cell biology resources

Brief understanding current key areas of cytoskeleton research

Brief understanding of current cytoskeleton research techniques

 

Page Links: Start Here | PubMed | Journals | NLM Online Textbooks | Online Mendelian Inheritence in Man

Start Here

1. We will divide the class into 3 separate groups.
2. Each group will investigate a specific filament system: microfilaments, intermediate filaments or microtubules.
3. Using online resources and search strategies we will investigate what the current key areas of research are on these cytoskeleton systems.
4. Some types of topics that can be identified are: research methods, components, tissue specific functions, motor molecules, associated disease topics, etc.
5. Capture data such as total number of results from specific searches, reference data, main finding of paper, technique used.
6. At the end of the class, your group will assemble a summary of what you have found, list your group members, and email to Dr Mark Hill (m.hill@unsw.edu.au) and your findings will be posted on this current page.

PubMed

National Center for Biotechnology Information (USA) NCBI "Established in 1988 as a national resource for molecular biology information, NCBI creates public databases, conducts research in computational biology, develops software tools for analyzing genome data, and disseminates biomedical information - all for the better understanding of molecular processes affecting human health and disease"

This is an external link, different databases can be selected from the pulldown menu, the selected option "Entrez" shows the results from searching a number of different NCBI databases simultaneously.

  Search Entrez NCBI Web Site ------------- PubMed Protein Nucleotide Structure Genome Books Domains 3D Domains Gene GEO GEO Datasets Journals LocusLink MeSH OMIM PMC PopSet SNP Taxonomy UniGene UniSTS for

Note the lefthand navigation panel on all UNSW Cell Biology pages include a link to this resource.

Journals

The Journal of Cell Biology

Nature

Science

Trends in Cell Biology

Public Library of Science - Biology

Proceedings of the National Academy of Sciences

Online Mendelian Inheritence in Man (OMIM)

  Search Entrez NCBI Web Site ------------- PubMed Protein Nucleotide Structure Genome Books Domains 3D Domains Gene GEO GEO Datasets Journals LocusLink MeSH OMIM PMC PopSet SNP Taxonomy UniGene UniSTS for

NCBI OMIM "This database is a catalog of human genes and genetic disorders authored and edited by Dr. Victor A. McKusick and his colleagues at Johns Hopkins and elsewhere, and developed for the World Wide Web by NCBI, the National Center for Biotechnology Information. The database contains textual information and references. It also contains copious links to MEDLINE and sequence records in the Entrez system, and links to additional related resources at NCBI and elsewhere."

NCBI OMIM Search Page

Medical Dictionary

This allows a Search of NIH Medlineplus Medical Dictionary. Type the word that you would like to find. If unsure of spelling, type the first few letters, followed by an asterisk(*).

Enter search term

Microtubules 1

Christine Yu

went to http://www.jcb.org/

To search through the content was redirected to Stanford University Highwire Press
Narrowed search to cytoskeletal substructures,
26 660 articles on microtubules
From Jan 2007 to Now there were 2887 articles

Microtubules offset growth site from the cell centre in fission yeast
Stefania Castagnetti, Béla Novák, and Paul Nurse
J. Cell Sci., Jul 2007; 120: 2205 - 2213.

• The research investigated the constraints governing the formation and positioning of new growth zones in the fission yeast cell.
• The positioning of growth zones was regulated by the polarity marker Tea1 delivered by microtubules; cells with short microtubules located the growth zone near the region where the microtubules terminated.
• A model was proposed for the activation of new growth zones; comprising a long-range laterally inhibitory component and a self-activating positive local component that is delivered to cell ends by Tea1 and the microtubules.
• The principle of this symmetry-breaking design may also apply to the morphogenesis of other cells.
• Axioplan Zeiss microscope for pictures.
• Nuclear displacement by centrifugation
• For microtubules cells were fixed in –80°C methanol for 1 hour, for standard Tea1 staining cells were fixed in –80°C methanol for 8 minutes
• Photographs were taken using a Deltavision microscope.
• Single microtubules were measured in individual cells after 30 minutes of MBC treatment and the longest considered as the microtubule length in that cell.
  

Disorganized Microtubules Underlie the Formation of Retraction Bulbs and the Failure of Axonal Regeneration
Ali Ertürk, Farida Hellal, Joana Enes, and Frank Bradke
J. Neurosci., Aug 2007; 27: 9169 - 9180.

Axons in the CNS do not regrow after injury, whereas lesioned axons in the peripheral nervous system (PNS) regenerate. Lesioned CNS axons form characteristic swellings at their tips known as retraction bulbs, which are the non-growing counterparts of growth cones.

The paper reported the analysis of the morphological and intracellular responses of injured axons in the CNS compared with those in the PNS. They found that retraction bulbs of injured CNS axons increase in size over time, whereas growth cones of injured PNS axons remain constant.
Retraction bulbs contain a disorganized microtubule network, whereas growth cones possess the typical bundling of microtubules.

Pharmacological disruption of microtubules in growth cones transforms them into retraction bulb-like structures whose growth is inhibited. Correspondingly, microtubule destabilization of sensory neurons in cell culture induces retraction bulb formation. Conversely, microtubule stabilization prevents the formation of retraction bulbs and decreases axonal degeneration in vivo. Finally, microtubule stabilization enhances the growth capacity of CNS neurons cultured on myelin. Thus, the stability and organization of microtubules define the fate of lesioned axonal stumps to become either advancing growth cones or nonrowing retraction bulbs.

Our data pinpoint microtubules as a key regulatory target for axonal regeneration.

Perfusion and sectioning: Animals were killed and perfused at the defined time points. In brief, animals were anesthetized with 5% chloral hydrate solution (prepared in saline) and perfused intracardially with 0.1 M PBS at a speed of 3 ml/min for 5 min, immediately followed by 4% paraformaldehyde (PFA) in PBS for 20 min for immunostainings and for 45 min for morphology analysis.

Immunohistochemistry: The primary antibodies used were antiglutamated (Glu)-tubulin (rabbit) at 1:500 dilution
In vivo imaging using an Olympus fluorescent stereomicroscope equipped with a 1_ Plan Apochromat objective and 1.6_ Plan Fluorite objective.

Microtubules 2

Microtubules

Belinda Lawrence

It appears that the current research relating to Microtubules does not focus on the microtubule structure and function directly, but employs microtubules to aid in understanding or research of other ideas. This suggests that the knowledge of microtubules is relatively sound, and thus confidence can be assumed in applying the knowledge of Microtubules to experimental and unknown areas.

Through a metasearch on Sirius I found this article from Nature. It talks about how bacterial pathogens operate by attacking intracellular pathways in their hosts. This paper discusses ideas of how pathogens target distinct cytoskeletal components, such as microtubules. It focuses on the polarity of Microtubules as a mechanism for the pathogens to enter or leave the host cell.

Insight

Nature 449, 827-834 (18 October 2007) | doi:10.1038/nature06247; Published online 17 October 2007

Review Article Manipulation of host-cell pathways by bacterial pathogens

Amit P. Bhavsar, Julian A. Guttman& B. Brett Finlay

Interactions of microtubules with effectors

• Microtubules are commonly targeted by microorganisms. These polarized structures are normally used for structural support and as tracks to guide and transport intracellular cargo, with the aid of microtubule-associated molecular-motor proteins.
• During certain infections, both the cargo transport and the microtubule assembly and/or disassembly dynamics can be modified and controlled by the pathogen. For example, on invasion by Shigella spp., the VirA protein interacts directly with heterodimers of -tubulin and -tubulin, promoting destabilization of the microtubules. This results in a localized absence of microtubules near the invading bacteria, thus aiding invasion by Shigella spp.
• A similar phenotype is seen during EPEC infections. In this case, localized microtubule depolymerization depends on the bacterial effector EspG, which, similarly to VirA, interacts directly with tubulin.
• Whereas Shigella spp. and pathogenic E. coli disassemble microtubules, a strain of Campylobacter jejuni has been shown to use microtubules and their associated molecular motors to aid invasion.
• Microtubules are polar structures that have distinct fast-growing (plus) and slow-growing (minus) ends, allowing the directional transport of cargo in cells. There are two general types of microtubule-based molecular motor found in the host-cell cytosol: kinesins and cytoplasmic dynein. Members of the kinesin superfamily generally transport cargo towards the plus ends of microtubules. By contrast, cytoplasmic dynein is thought to be a minus-end-directed motor.
• Microtubules, and particularly dynein, have been implicated in invasion by a strain of C. jejun. Given that the minus ends of microtubules in cultured, non-polarized cells are directed towards the interior of the cell, this model seems plausible.
• However, in polarized cells (such as those present in intestinal epithelial barriers), microtubule polarity is reversed, so dynein would not transport C. jejuni towards the cell interior. Therefore, further investigation into the uptake of C. jejuni by host cells is required.

Microtubules 3

Jackie Hara-Crockford

Lorch DP, Lindemann CB, Hunt AJ.The motor activity of mammalian axonemal dynein studied in situ on doublet microtubules.Cell Motil Cytoskeleton. 2008 Apr 17; [Epub ahead of print]

• These results reveal fundamental properties underlying cooperative dynein activity in flagella, differences between mammalian and non-mammalian flagellar dynein, and establish the use of natural tracks of dynein arranged in situ on the doublet microtubules of bovine sperm as a system to explore the mechanics of the dynein-microtubule interactions in mammalian flagella. Cell Motil. Cytoskeleton 2008. (c) 2008 Wiley-Liss, Inc. This was done by an in situ assay in which polymerized single microtubules glide along doublet microtubules extruded from disintegrated bovine sperm flagella at a pH of 7.8.

Schatten H.The mammalian centrosome and its functional significance. Histochem Cell Biol. 2008 Apr 24

• This review highlights the centrosome's significant roles in cell cycle events in somatic and reproductive cells and discusses centrosome abnormalities and implications in disease.

Hughes JR, Meireles AM, Fisher KH, Garcia A, Antrobus PR, Wainman A, Zitzmann N, Deane C, Ohkura H, Wakefield JG.A Microtubule Interactome: Complexes with Roles in Cell Cycle and Mitosis.

• This study therefore demonstrates that biologically relevant data can be harvested using such a multidisciplinary approach, and identifies new MAPs, many of which appear to be important in cell division. This study used a MT cosedimentation assay, combined with 1D and 2D PAGE and mass spectrometry, to identify over 250 MAPs from early Drosophila embryos

Jordan MA, Kamath K.How do microtubule-targeted drugs work? An overview. Curr Cancer Drug Targets. 2007 Dec;7(8):730-42. Review. PMID: 18220533 [PubMed - indexed for MEDLINE]

• The importance of microtubules in mitosis makes them a superb target for a group of highly successful, chemically diverse anticancer drugs. This overview covers microtubule assembly dynamics, the exquisite regulation of microtubule dynamics in cells by endogenous regulators, the important role of microtubule dynamics in mitosis, the diversity and number of microtubule-targeted drugs undergoing clinical development, the antimitotic mechanisms of microtubule-targeted drugs with emphasis on suppression of microtubule dynamics by vinblastine and taxol, the role of drug uptake and retention in the efficacy of microtubule-targeted drugs, and the anti-angiogenic and vascular-disrupting mechanisms of microtubule targeted drugs.

Sakamoto T, Uezu A, Kawauchi S, Kuramoto T, Makino K, Umeda K, Araki N, Baba H, Nakanishi H.Mass spectrometric analysis of microtubule co-sedimented proteins from rat brain. Genes Cells. 2008 Apr;13(4):295-312. PMID: 18363962 [PubMed - indexed for MEDLINE] -

• This analysis provides an extensive list of potential candidates for future study of the molecular mechanisms of MT-based functions and structures

Microtubules 4

3187491 3185666 3188178

Brouhard GJ.,  Stear GH., Noetzel TL., Al-Bassam J., Kinoshita K., Harrison SC.,

Howard J., and Hyman A.A., 2008 XMAP215 Is a Processive Microtubule Polymerase. Cell.132 (1): 79-88

• This article was located on the Sciendirect database through Sirius and is within the journal CELL.

• This is an article about a specific protein thought to be associated with the structure and formation of microtubules.  Microtubules are extended by the addition and removal of tubulin subunits at the ends of the microtubule. Cell proliferation and differentiation requires this process to occur rapidly for the remodelling the cytoskeleton.  XAMP215 is the protein that is thought to allow this rapid remodelling and growth.

Abstract:

“To determine how XMAP215 accelerates growth, we developed a single-molecule assay to visualize directly XMAP215-GFP interacting with dynamic microtubules. XMAP215 binds free tubulin in a 1:1 complex that interacts with the microtubule lattice and targets the ends by a diffusion facilitated mechanism. XMAP215 persists at the plus end for many rounds of tubulin subunit addition in a form of ‘‘tip tracking.’’ These results show that XMAP215 is a processive polymerase that directly catalyses the addition of up to 25 tubulin dimers to the growing plus end. Under some circumstances XMAP215 can also catalyse the reverse reaction, namely microtubule shrinkage. The similarities between XMAP215 and formins, actin polymerases, suggest that processive tip tracking is a common mechanism for stimulating the growth of cytoskeletal polymers.”

• The article describes the growth rates of microtubulin in different species and tries to answer the question of how the specific protein promotes microtubule growth.  One issue brought up was “Acceleration of growth could result from rapid cycling onto and off the microtubule end or from multiple tubulins being brought at once”.   The proteins bind tubulin subunits in solution and take them to the extending micro tubule. The article firstly describes XAMP215 as a tubulin shuttle, but through microscopic assays of the microtubule tubule formation, the article determined that “XMAP215 does not, in fact, act as a tubulin shuttle. Rather, XMAP215 acts as a processive polymerizing enzyme or polymerase: XMAP215 forms a 1:1 complex with tubulin and resides for long periods at microtubule plus ends, where it catalyses repeated rounds of addition of tubulin into the microtubule polymer.”

Microtubules 5

Kim Nair

Schmidt M, Bastians H. (2007) Mitotic drug targets and the development of novel anti-mitotic anticancer drugs. Drug Resistance Updates 10 (4-5): 162 – 181.

Link:

http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6WDK-4P9TB0F-1-Y&_cdi=6769&_user=37161&_orig=search&_coverDate=10%2F31%2F2007&_sk=999899995&view=c&wchp=dGLbVtz-zSkzS&md5=132217585ae4ab5051219b6eacba9171&ie=/sdarticle.pdf

Microtubule binding anti-cancer drugs

• many chemical compounds targeting microtubules, mostly derived from natural sources,

exert their primary mode of action on proliferating tumor cells by a blockade of mitosis, which subsequently leads to the induction of cell death

• microtubule binding drugs frequently referred to as spindle poisons
• bind to and inhibit the function of microtubules of

the mitotic spindle apparatus

• stop of the cell cycle in mitosis and subsequently to the induction of

tumor cell death

• functional inhibition of
• KSP/Eg5 activates the mitotic spindle checkpoint leading to a cell cycle arrest in mitosis.
• mitotic kinesins are well druggable targets]

Anti-cancer drugs targeting KSP/Eg5

• Cytokinetics has been the leader in the development of KSP/Eg5 kinesin inhibitors
• KSP/Eg5 inhibitors activate the mitotic spindle checkpoint  and cells arrest in mitosis with monoastral spindles.

Verrills NM, Liem NL,  Liaw TY, Hood BD, Lock RB, Kavallaris M. (2006).

Proteomic analysis reveals a novel role for the actin cytoskeleton in vincristine resistant childhood leukemia – An in vivo study

Proteomics6: 1681–1694.

• antimicrotubule agent used in the treatment of childhood acute lymphoblastic leukemia (ALL)
• Vincristine (VCR) is an antimicrotubule agent used in the induction and maintenance therapy of childhood ALL
• VCR binds to the b-tubulin subunit of a/b-tubulin heterodimers and causes depolymerization of microtubules. As microtubules are crucial for cell division, they are effective targets for anticancer therapy
• Proteomic analysis, utilizing high-resolution 2-DE coupled with MS, is a powerful means to identify

differential protein expression between biological samples.

Intermediate Filament 1

Elizabeth Garay, Rita Muscat, Rahela Waseeq

Biological abstracts search word : intermediate filament = 141 papers in 2007
6647 in any year

Research findings in the last year:

1. Intermediate filament protein Nestin identifies stem/ progenitor cells in
adult tissues and its function in common lethal cancers
Nestin mRNA was detected in cell lines from small cell lung, and breast
cancers, and particularly elevated in cell lines derived from prostate cancer
metastases.

ref: Kleeberger, Wolfram [Author]; Bova, G. Steven [Author]; Nielsen, Matthew
E. [Author]; Herawi, Mehsati [Author]; Chuang, Ai-Ying [Author]; Epstein,
Jonathan I. [Author]; Berman, David M. [Author, Reprint Author; E-mail:
berman@jhmi.edu]. Roles for the stem cell-associated intermediate filament
nestin in prostate cancer migration and metastasis [Article] Cancer Research. 67
(19). OCT 1 2007. 9199-9206.

2. Examine IF involvement, along with cytolinker participation, in sequential
caspase-mediated protein cleavages that are part of the overall cell death
execution, particularly those that generate new functional IF protein fragments
and uncover neoantigen markers. Finally, we report on the usefulness of these
markers as diagnostic tools for disease-related aspects of apoptosis in humans

ref:http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WFC-4NG3TCW-
1&_user=37161&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000004218&_versio
n=1&_urlVersion=0&_userid=37161&md5=c7950b49091104ed128fca5c2ed5f85e

3. The idea of IFs as “signaling platforms” (e.g.,see Paramio and Jorcano 2002;
Coulombe and Wong 2004; Pallari and Eriksson 2006) has officially emerged,and
further research is needed to uncover the extent ofthis phenomenon and how it
works.

Paramio, J.M. and Jorcano, J.L. 2002. Beyond structure: Do intermediate
filaments modulate signalling? Bioessays 24:836–844.

Pallari, H.M. and Eriksson, J.E. 2006. Intermediate filaments as signaling
platforms. Sci. STKE 366: 53.

Research techniques:

Electroporator

Ref: http://biology.plosjournals.org/perlserv/?request=get-
document&doi=10.1371/journal.pbio.0050233

Intermediate Filament 2

Search Intermediate Filaments:

Total journals found 2390 in Medline.

Journals published in 2008 are 127.

Journals published in 2007 are 707.

Journals published in 2006 are 398.

Molecular evolution of type VI intermediate filament proteins

 

This research paper talks about the three main proteins of type IV intermediate filament. These proteins are Tanabin, transitin and nestin. Tanabin are important in development in frogs, whereas transitin is in birds and the last one nestin related to mammals. Another type IV intermediate filament is called synemin is expressed in undifferentiated and mature muscle cells of birds and mammals.

The main results of this research paper shows that tanabin, transitin and nestin genes share intron positions and sequence identities. Phylogenic analyses show these have a similar chromosomal context and display. The appearance of repeated motifs is brought about by fast evolution rates of C- terminal extremity.

 The major finding of this article is the  rapid evolution of the C-terminal extremity of nestin orthologs could be responsible for their different functions of type IV intermediate filaments.

Reference:

Guérette, D, Khan P.A, Savard E.P, and Vincent,M. Molecular evolution of type VI intermediate filament proteins (2007), Published in BMC Evolutionary Biology, Volume 7, page 164.

By

Puourach Thomas  Z3201620

Amina Razzaq   Z3162323

Ngatho Mugo.  Z3161544

Microfilaments!

Salwan Abbas, Gab Tan, Sarah Benjamin, Saranya Madana Gopal

Searching for “microfilament”- 15599 sources

Limit to last year- 820 sources

1-

Darin McDonald, Gustavo Carrero, Christi Andrin, Gerda de Vries, and Michael J. Hendzel
Nucleoplasmic ß-actin exists in a dynamic equilibrium between low-mobility polymeric species and rapidly diffusing populations
J.Cell Biol., Feb 2006; 172: 541 - 552.


Major findings - β-Actin, once thought to be an exclusively cytoplasmic
protein, is now known to have important functions within the nucleus. Our results indicate that actin and actin-containing complexes are reduced in their mobility through the nucleoplasm diffusing at 0.5 μm2 s−1.

Technique - We define the dynamic properties of nuclear actin molecules
using fluorescence recovery after photobleaching.

2-

Storch, Taatjes, Bouffard, Locknar, Bishop, Langevin, Alpha smooth muscle actin distribution in cytoplasm and nuclear invaginations of connective tissue fibroblasts. [Journal Article. Research Support, N.I.H., Extramural] Histochemistry & Cell Biology. 127(5):523-30, 2007

Major finding: These alpha-SMA were found close to the nuclear membrane but not extending within nucleoplasm. The authors think that alpha-SMA might have a role in “cellular and nuclear mechanotransduction as well as nuclear transport”.

Techniques used immunohistochemistry and confocal scanning laser and electron microscopy to investigate the “structural organisation of alpha smooth muscle actin”.