Jason C. Steel, George F. Atweh, Charmaine A. Ramlogan-Steel The Identification of Stathmin-like Proteins Essential for Microtubule Functions in Stathmin Knockout Mice Mahima Venkatesh Jason C. Steel, George F. Atweh, Charmaine A. Ramlogan-Steel

What is stathmin? First identified in 1983 by Feurestein and Cooper Approx 17kDa Pseudonyms include p17, p18, p19, op18, lap18, oncoprotein 18, prosolin, metablastin

Roles of Stathmin Regulation of the cell cycle-important in mitosis and microtubule dynamics. It is necessary for the formation of the mitotic spindle which implies that its deficiency can lead to problems with cell division

The role of stathmin in microtubule formation/degradation Stathmin regulates microtubule dynamics by promoting depolymerization of microtubules and/or preventing polymerization of tubulin heterodimers Stathmin binds two alpha/beta tubulin heterodimers through a helical C-terminal region. The N-terminus of stathmin caps the alpha1-tubulin to prevent assembly into microtubules.  Model for the role of stathmin in the regulation of microtubule dynamics. Microtubules (MT) continuously switch between phases of polymerization and depolymerization. Stathmin can sequester unpolymerized tubulin by binding two α/β‐tubulin heterodimers (represented by light and dark shaded circles), thus diminishing the pool of tubulin heterodimers available for polymerization. Stathmin can also bind to the end of polymerized microtubules and increase the rate of catastrophe by inducing a conformational change that promotes microtubule depolymerization. Iancu Rubin, C and Atweh, GF. The role of stathmin in the regulation of the cell cycle. Journal of Cellular Biochemistry Volume 93, Issue 2, pages 242–250, 1 October 2004 4

The role of stathmin in the cell cycle Iancu Rubin, C and Atweh, GF. The role of stathmin in the regulation of the cell cycle. Journal of Cellular Biochemistry Volume 93, Issue 2, pages 242–250, 1 October 2004

Stathmin expression in cancer Stathmin overexpression has been observed in many cancers including prostate, breast and ovarian carcinomas and acute leukemia. Increased rate of microtubule turnover is thought to contribute to the characteristic change in cell shape associated with malignant cancer cells Higher levels of stathmin correlate with more aggressive disease and poorer prognosis. Yuan et al. showed that stathmin is associated with tumor progression and early recurrence in patients with HCC. Pull some key figures from stathmin + cancer (Put my invitro data in) Chemo + anti-stathmin

Stathmin Knockout Mice Given these important functions it was thought that the development of a stathmin knockout (KO) mouse would be impossible. Schubart et al., 1996- developed the stathmin -/- mouse. Initially, KO mouse was phenotypically normal.

Stathmin KO Mouse Liedtke et al. (2001): stathmin−/− mice showed a progressive PNS and CNS axonopathy with structural damage and associated functional impairment. Martel et al.: deficiency in fear and Parental care affected in females Zahedi et al. (2006): IRI in KO and WT mice and found that KO mice had delayed tubular repair and developed more tubular fibrosis when compared to WT littermates. The same study showed increased expression of SCLIP, a stathmin-related protein, in the nervous system of the aging mice. In another line of investigation, the suppression of the only homologue of the vertebrate stathmin family in Drosophila led to dramatic impairment of germ cell migration and severe anomalies in the development of nervous system [Ozon et al., 2002]. The remarkable lack of phenotype in the stathmin null mice and the severe impairment of development in an organism with a single stathmin-likegenesuggestsafunctionalredun- dancy among stathmin family members in vertebrates. This redundancy is supported by a recent study that assessed the expression of stathmin and its other related proteins by quantitative RT-PCR analysis in a variety of human tissues [Bieche et al., 2003]. This study showed that the pattern of expression of the SCLIP member of the stathmin-like family, like the expression of stathmin itself, is ubiquitous, suggesting that stathmin and SCLIP may be functionally redundant.

Weight comparison in WT vs KO mice Age (weeks) 3 12 Weight comparison in WT vs KO mice Comparison of WT vs KO Bone Marrow Cell Count Bone marrow cell counts (107)/mouse

Objective The development of stathmin KO mice and their limited phenotypic abnormalities suggest that other proteins may be compensating for stathmin’s role in microtubule dynamics. Our project’s goal is to identify the stathmin-like proteins or molecules which appear to compensate for the loss of stathmin.

Methods Examined the expression of 18 stathmin-like proteins from cells derived from KO mice and wild-type mice Harvested bone marrow and splenocytes from these mice and differentiated pre-B, megakaryocytes and granulocyte/macrophages cells using different culture conditions.

Methods Isolated total RNA from cells and performed reverse transcriptase reactions to generate cDNA Using primers specific for the 18 genes, quantitative PCR reactions were performed to determine whether any of the genes were up- or down-regulated in the KO mice The identification of these proteins may provide insight into the essential molecular interactions required for microtubule functions.

Genes Stathmin 1-4 Clasp 1 Clasp 2, Clip1, Clip 2, Macf1, Mtap4, Mapre 1, Mapre 2, Mapt, Mid1, Racgap, Phldb2, Spastin, Katanin1, Kif2c B-Actin

Katna1 is up-regulated in Stathmin KO Mice shows changes in relative gene expression between WT and KO mice. The bars above the x-axis shows genes that are upregulated in KO mice compared to WT and the bars under the x-axis show genes that are downregulated in the KO mice when compared to WT mice. From the graph, you can see that there is a an upregulation of Katanin (Katna1) and to a lesser extent, an upregulation in Clip2. There are also downregulations in Mapre 1, Macf1 and Spastin. 

Results Summary These results represent 5 separate qPCR experiments From the graph, you can see that there is a an upregulation of Katanin (Katna1) and to a lesser extent, an upregulation in Clip2. There are also downregulations in Mapre 1, Macf1 and Spastin. 

Katanin Results imply that it may be compensating for loss of stathmin Function: releases microtubules from their attachment to a microtubule depolymerization observed at the poled of spindles during meiosis and mitosis (Taylor et al p.1000) May be involved in microtubule release and depolymerizaion in proliferating cells in interphase (Taylor et al p.1000) Katanin is a heterodimer consisting of a 60-kDa microtubule-stimulated ATPase that requires ATP hydrolysis to disassemble microtubules, and an 80-kDa subunit that targets the complex to centrosomes and regulates the microtubule- severing activity of the p60 subunit

http://www.hindawi.com/isrn/mb/2012/596289/fig2/

Clip 2 Results imply that it may be compensating for loss of stathmin The protein encoded by this gene belongs to the family of cytoplasmic linker proteins proposed to mediate the interaction between specific membranous organelles and microtubules. Associated with both microtubules and an organelle called the dendritic lamellar body.

Clip2 Function: Seems to link microtubules to dendritic lamellar body (DLB), a membranous organelle predominantly present inbulbous dendritic appendages of neurons linked by dendrodendritic gap junctions. May operates in the control ofbrain-specific organelle translocatio

Future Studies Confirm expression of katanin in the KO mice with western blot Looking at the effects of stathmin KO combined with katanin knockdown

References Iancu Rubin, C and Atweh, GF. The role of stathmin in the regulation of the cell cycle. Journal of Cellular Biochemistry Volume 93, Issue 2, pages 242–250, 1 October 2004 Schubart UK, Yu J, Amat JA, Wang Z, Hoffmann MK, Edelman W. 1996. Normal Development of mice lacking matablastin (P19): A phosphoprotein implicated in cell-cycle regulation. J Biol Chem 271:14062–14066. Liedtke W, Leman EE, Fyffe REW, Raine CS, Schubart U. 2002. Stahmin-deficient mice develop an age-dependent axonopathy of the central and peripheral nervous system. Am J Pathol 160:469–480. Martel et al. 2012 Murine GRPR and stathmin control in opposite directions both cued fear extinction and neural activities of the amygdala and prefrontal cortex. Taylor et al. The Evolution of Brain and Behaviour. Garland Science, 2010 Zahedi et al. 2006 Stathmin-deficient mice develop fibrosis and show delayed recovery from ischemic-reperfusion injury.

Acknowledgements Charmaine Ramlogan-Steel, M.D. Jason Steel, Ph.D George Atweh, M.D. Brittany and everyone else from the Atweh Lab

Thank you! Questions?