Določanje vpliva plektina na razporeditev proteina tau in dinamiko mikrotubulov v mišjih astrocitih s superločljivostno mikroskopijo STED in konfokalno mikroskopijo

Kovačić, Marko (avtor), Jorgačevski, Jernej (mentor)

Astrociti imajo številne funkcije, s katerimi vzdržujejo homeostazo v centralnem živčnem sistemu. Pomemben povezovalni protein elementov citoskeleta v astrocitih je plektin, večfunkcionalni citolinkerski protein iz družine plakinov. Ker je malo znanega o vplivu plektina na dinamične lastnosti mikrotubulov in lokalizacijo proteina tau v astrocitih, smo to želeli preveriti v imortaliziranih mišjih astrocitih, ki izražajo plektin (Plec+/+p53-/-) in ki ne izražajo plektina (Plec-/-p53-/-). Dinamiko mikrotubulov smo spremljali s konfokalno fluorescenčno mikroskopijo živih celic, pri čemer smo rastoče konce mikrotubulov označili s proteinom Eb3, sklopljenim z zelenim fluorescenčnim proteinom. Ugotovili smo, da mikrotubuli v odsotnosti plektina polimerizirajo hitreje. Ugotovili smo, da imajo mikrotubuli v odsotnosti plektina večji koeficient usmerjenosti polimerizacije, bolj radialno usmerjeno rast in manjšo kotno disperzijo rasti glede na centroid celice. Plektin ni imel vpliva na število iniciacij rasti novih mikrotubulov in na gostoto rastočih mikrotubulov v celicah. Ugotovili smo, da so mikrotubuli v odsotnosti plektina manj fragmentirani, bolj prepleteni in imajo večjo celokupno dolžino, normirano na ploščino celice. Tako v mišjih astrocitih Plec+/+p53-/- kot tudi v mišjih astrocitih Plec-/-p53-/- smo ugotovili relativno visoko stopnjo izražanja proteina tau, ki se je pojavljal v skupkih. Kvalitativno nismo zaznali, da bi skupki proteina tau kolokalizirali z mikrotubuli. Ugotovili smo, da protein tau v odsotnosti plektina zaseda večji delež imortaliziranih mišjih astrocitov in da se večji delež proteina tau nahaja v perifernem delu celic v primerjavi z astrociti, ki izražajo plektin. S superločljivostno mikroskopijo z vzbujenim praznjenjem emisije (STED) smo ugotovili, da so skupki proteina tau gostejši v odsotnosti plektina. Omenjeni fenotip smo poskusili rešiti z lipofekcijo astrocitov Plec-/-p53-/-, in sicer z vnosom zapisa za izoobliko plektina P1c. Transfekcija je bila uspešna, vendar med transficiranimi in netransficiranimi celicami ni bilo razlike v gostoti in lokalizaciji skupkov proteina tau. Ugotovili smo, da je postopek lipofekcije bistveno znižal delež in gostoto skupkov proteina tau ter spremenil njihovo lokalizacijo tako v transficiranih kot v netransficiranih celicah v primerjavi z mišjimi astrociti v eksperimentu brez lipofekcije. Naši rezultati nakazujejo, da plektin vpliva na lastnosti mikrotubulov in razporeditev proteina tau v imortaliziranih mišjih astrocitih. V nadaljevanju bi bilo treba preveriti, kakšen je mehanizem vpliva plektina na omenjene lastnosti in kaj so razlogi, da se protein tau nahaja v obliki skupkov v imortaliziranih mišjih astrocitih. Potrebno bi bilo preučiti vpliv plektina na dinamiko mikrotubulov in razporeditev proteina tau tudi v primarnih astrocitih.

• Jezik: Slovenski jezik
• Ključne besede: imortalizirani mišji astrociti, plektin, mikrotubuli, protein tau, STED, konfokalna mikroskopija
• Vrsta gradiva: Magistrsko delo/naloga
• Tipologija: 2.09 - Magistrsko delo
• Organizacija: FKKT - Fakulteta za kemijo in kemijsko tehnologijo
• Leto izida: 2026
• PID: 20.500.12556/RUL-184437
• COBISS.SI-ID: 288461827
• Datum objave v RUL: 07.07.2026

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Determining the role of plectin on tau protein distribution and microtubule dynamics in mouse astrocytes using superresolution STED microscopy and confocal microscopy

Izvleček

Astrocytes have many functions that maintain homeostasis in the central nervous system. An important connecting protein of the cytoskeleton elements in astrocytes is plectin, a multifunctional cytolinker protein from the plakin family. Since little is known about the influence of plectin on the dynamic properties of microtubules and the localization of tau protein in astrocytes, we wanted to verify this on immortalized mouse astrocytes, which express plectin (Plec+/+p53-/-) and which do not express plectin (Plec-/-p53-/-). Microtubule dynamics was monitored by live-cell confocal fluorescence microscopy, where the growing ends of microtubules were labeled with the protein Eb3 coupled to green fluorescent protein. We showed that microtubules polymerize faster in the absence of plectin. We also found that microtubules in the absence of plectin have a higher orientation coefficient of polymerization, more radially oriented growth relative to the cell centroid, and lower angular dispersion of growth. Plectin had no effect on the rate of initiation of growth of new microtubules and on the density of growing microtubules in cells. We found that microtubules in the absence of plectin are less fragmented, more overlapped, and have a higher total length normalized to the cell area. We found a relatively high level of tau protein expression in the form of aggregates in both Plec+/+p53-/- and Plec-/-p53-/- mouse astrocytes. We did not qualitatively detect that tau protein aggregates colocalized with microtubules. We showed that tau protein occupies a larger proportion of immortalized mouse astrocytes in the absence of plectin and that more tau protein is located in the peripheral part of the cells compared to astrocytes, which express plectin. We showed using STED superresolution microscopy that tau protein aggregates are denser in the absence of plectin. We attempted to rescue the aforementioned phenotype by lipofection of Plec-/-p53-/- astrocytes with a gene for the plectin isoform P1c. The transfection was successful, but there was no difference in the density and localization of tau protein aggregates between transfected and untransfected cells. We found that lipofection procedure alone significantly reduced the area and density of tau protein aggregates and changed their localization in both transfected and untransfected cells compared to mouse astrocytes in the experiment wihout lipofection. Our results suggest that plectin affects microtubule properties and tau protein distribution in immortalized mouse astrocytes. In the future, it would be necessary to investigate the mechanism of plectin's influence on the aforementioned properties and what are the reasons for tau protein to be found in the form of aggregates in immortalized mouse astrocytes. It would be necessary to study the influence of plectin on microtubule dynamics and tau protein distribution in primary astrocytes as well.

• Ključne besede: immortalized mouse astrocytes, plectin, microtubules, tau protein, STED, confocal microscopy