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1 The College at Brockport: State University of New York Digital Biology Master s Theses Department of Biology Regulation of Actin Dynamics by Melanin- Concentrating Hormone Potentiates Downstrean Signaling Extracellular Signal-Regulated Kinase in Cultured Cells Scott Michael Portwood The College at Brockport Follow this and additional works at: Part of the Biology Commons Reposiry Citation Portwood, Scott Michael, "Regulation of Actin Dynamics by Melanin-Concentrating Hormone Potentiates Downstrean Signaling Extracellular Signal-Regulated Kinase in Cultured Cells" (2011). Biology Master s Theses This Thesis is brought you for free and open access by the Department of Biology at Digital It has been accepted for inclusion in Biology Master s Theses by an authorized administrar of Digital For more information, please contact kmyers@brockport.edu.

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3 DEFENSE NOT APPROVED MASTER'S DEGREE ADVISORY COMMITTEE "5/JRj_ // Date Committee Member I I Date 3/;rju 7 Dat Chairman, Dept. of Biological Sciences

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34 A. B. c. D. NoMCH E. MCH +2min F. MCH +5min MCH +lomin llntrcated 3T3-U Prcadipocytcs 3T3-U Prc-adipocytes treated with 1 µm MCH MCH Addition 31'3-Ll l're--ad1nocvtes Untreated 3T3-L I cells were serum starved and A,E. left untreated or treated with MCH for B. minutes minutes or D. I 0 minutes. Actin filaments were stained with Alexa-Fluor Phalloidin and DNA was stained with DAPL and l OOX and with a Axiocam MRm fluorescence software.

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36 MCH 'nun Fibers w/ few Stress Fi hers Cell 24 32± 3,86 0 I) 98:i: 0.'2"o ± (P 0.05) 5"' : (P (J.()02:') (P O.ll023J 18 0 l Oli±O B 0 o were serum starved for l hour followed by a time course addition of I ttm MCH. Actin filaments were stained with Alexa-Fluor Phalloidin and DNA was stained with DAPL fluorescence cells were as I) membrane extensions, or small and round in blinded a T~test untreated cells in the same category. or were cons[dered stat1st1cally tal of cells in each category were were was

37 was apparent 1 a a star at 5 10 minutes e so Intermediate I Small Round Statistically Significant MCH. Actin fluorescence m1c:roscwly, cells a a

38 a ± an exaggerated effect on be () I Fibroblast-like Small Round * Statistically Significant signaled by MCHR J or l Omin. Using fluorescence rn11~ro:~conv. were categorij:cd as I) having prominent actin stress fibers, being round with many membrane extensions, or 3) small and round in blinded experiments. Statistical was using T~test as compared untreated the same scoring the 95i1i or higher were considered statistically signific<mt. A tal of 4 ncr rrnc:cl and approximately 200 cells in each category are reported.

39 act treatments and subsequent washes. When the intermediate cell was no A. PLC Inhibir No Rx 2min Smin lomin Small Round mediated 3T3-Ll pre--adipocytes were pre-treated with known PLC inhibir U- 73 determine if MCHR l re4uires activation via the CJq Protein pathway signal actin morphology rearrangement. Cells on poly lysine-coated slides were serum starved for J h, then pretreated with 2µM ICillowed by I ~tm MCH for 5, or I 0 min. Using fluoresct:nce microscopy, cells were categorized as I) having prominent actin stress fibers, 2) being round with many plasma membrane extensions, or 3) being small and round in blinded experiments. Two experiments were performed and approximately 200 eel Is for each time point were analyzed in each experiment Averages range are reported.

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41 A. B No Rx MCHR-1 MCH PMC-3881-PI MCH + PMC-3881-PI Treatment I Intermediate I Small Round Statistically Significant inhibits MCH-Mediated Actin Ke,11rr~m~1~m~~nts in 3T3-Ll pre- MCHR I. MCHR2 or both. MCH. I neither was seen

42 A) B) C) 9. imaged Chinese Hamster Ovary Axiocam MRrn fluorescence microscope with AxioView imaging software. MCHR l were pre-treated with I OpM Latrunculin and were run on a lo'vo SDS-PAGE gel and western blot was nr rtnr nw lf using l: 1000 rabbit P-MAPK, I mouse anti-total p42/44 MA PK antibodies. fhe blot shows Latrunculin activates MAPK without MCH addition (-j> ).

43 Cychalasin confirmed by inhibit actin pre-adipocytes were 40-50% 1 (Figure consisting of numerous stress lob) a stress A) B) Untreated loµm A) 3TJ-L l Pre-adipocyte cells serum starved for l hour, pre-treated with I µm Cychalasin D for 30 min, followed by exposure I µ.m MCH. Actin filaments were stained with Alexa-Fluor Phalloidin and DNA was stained with DAPJ. Cells were imaged 40X and captured with a MRm fluorescence microscope with Axio View imaging softvvare.

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45 a A} No Treatment MCH (min) TOTAlMAPK PHOSPO-MAPK B) No f\xn MCH MCH MCH +Smin <-lomin +30min Iii Untreated iflj Cychalasin D Densimetry performed with Adobe Phoshop Figure Pharmacological Agent Cychalasin D Prolongs MAPK activation in stably expressing MCHRJ Representative performed where Chinese hamster tagged MCHR l were serum for I hour, remained untreated, or pre-treated time course addition of l µm MCH. The samples were run on a I 0% SDS-PAGE gel and a western blot was performed using I I 000 rabbit anti-p42/44 P-MAPK, I: I 000 mouse anti-total p42/44 MAPK antibodies. Equal volumes of samples were loaded and measured against the tal MAPK antibody (Total MAPK) and phosphorylated MAPK present (Phospho-MAPK). Both A&B untreated CHO cells show activation at min, fo!!owed by in MA.PK activation at l 0 and 30 minutes. Cychalasin D treated cells show MAPK activation at S minutes, followed by only a slight decrease in MAPK activation. Densimetry was performed using Adobe Phoshop. was

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47 A) 3T3~l1 B) MCH TOTAlMAPK ~ PHOSPO MAPK ~ V c 1.00 :J.E _a ~ I I No Rxn MCH MCH MCH MCH +Sm in + 10rnin +20min +30min Ill MAPI< Activation Densimetry performed with Adobe Phoshop Mgnalmg Can be Measured by MAPK Activation in 3T3-U Pre-:ulmocvt.es nre-aamocvt1es were serum starved for I hour followed by a time course addition of were run on a I 0% SOS-PAGE and a western blot was using 1: 1000 rabbit u11c.1,.,-,~, -,-T I: 1000 mouse anti-total MAPK antibodies. volumes were loaded and measured the tal MAPK and MAPK present MCH addition resulted in MAPK activation at 5 minutes, followed by a successive decrease in activation at 10, 20, and 30 minutes. B. was over

48 1 A) D) Undifferentiated 3T3-L1 Differentiated 3T3-L1 Nmt ;rn~1mn lipid had visual oil red filaments were stained with Alexa-Fluor Phalloidin and DNA was stained with DAPI. was successful. B & Actin F.Cells in the 6crn dishes 1

49 were on I a western mouse was IS A) Undifferentiated Differentiated MCH (min) lo 30 B) moo No Rxn MCH +5rnin MCH tlornin MCH +30rnin!Iii Unditterentiated '!Iii Differentiated Densimetry performed with Adobe Phoshop Representative experiments perfrxmed where 3T3-L I ore:-acilrn)cvtes differentiated from pre-adipocytes adipocytes followed by a time course of l ~tm MCH. Samples were run on 10% SOS-PAGE gel and a blot was performed l :1000 rabbit anti-p42/44 P-MAPK, I: 1000 mouse anti-total p42/44 MAPK antibodies. Equal volumes of samples were loaded and measured tal MAPK antibody (Total MAPK) and MAPK present (Phospho-MAPK). Higher MAPK is seen in undifferentiated adipocytes compared differentiated adipocytes. Densimetry was performed using Adobe Phoshop

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51 MCHR1 Internalization Time in minutes Linked lmmmiosorbent Verifies Loss of MCHRl ext)ressu12 MCHR 1 were grown 80% confluence in a 12-well dish. Cells were mouse I MCH for 1 Omin, and cells fixed with 4% Rabbit anti-mouse was added and cells were POD blue, followed by sulfuric acid sp the substrate reaction. Plates were MCH treated cells lose MCHR 1 surface over time. This verifies the assay is not an

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