ITSN1 and Ruk/CIN85 colocalized to clathrin-coated pits in MCF-7 cells
Activation of receptor tyrosine kinases (RTK) by corresponding ligands results not only in signal propagation, but also initiates a number of processes, such as clathrin-mediated endocytosis, which precisely regulate biological outcome. These processes are tightly controlled by coordinated action of...
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Інститут молекулярної біології і генетики НАН України
2009
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nasplib_isofts_kiev_ua-123456789-1529642025-02-23T17:26:04Z ITSN1 and Ruk/CIN85 colocalized to clathrin-coated pits in MCF-7 cells Інтерсектин 1 та Ruk/CIN85 колокалізуються в клатрин-облямованих ямках клітин лінії MCF-7 Интерсектин 1 и Ruk/CIN85 колокализуются в клатрин-окаймленных ямках клеток линии MCF-7 Nikolaienko, O.V. Skrypkina, I.Ya. Tsyba, L.O. Drobot, L.B. Rynditch, A.V. Короткі повідомлення Activation of receptor tyrosine kinases (RTK) by corresponding ligands results not only in signal propagation, but also initiates a number of processes, such as clathrin-mediated endocytosis, which precisely regulate biological outcome. These processes are tightly controlled by coordinated action of a plethora of proteins – enzymes, scaffolds and inhibitory molecules. An example of an endocytic accessory protein that also functions in cell signaling is provided by intersectin 1 (ITSN1). Previously we have shown that ITSN1 forms a complex with adaptor protein Ruk/CIN85 and ubiquitin ligase Cbl-b, which are implicated in down regulation of RTK. The present study aimed to determine the subcellular localization of ITSN1-Ruk/CIN85 complexes relatively to clathrin light chain and Cbl-b. Methods. Transient transfection of MCF-7 breast adenocarcinoma cells with the constructs containing Omni-tagged intersectin 1 and clathrin light chain fused with mCherry fluorescent protein was utilized to determine subcellular localization by direct or indirect immunofluorescence. Results. We found that Ruk/CIN85-ITSN1 complexes partially colocalized with Cbl-b and clathrin light chain in MCF-7 cells. Conclusions. In our report we provide experimental evidence that ITSN1-Ruk/CIN85 complexes exist in pre-assembled state with Cbl-b and are targeted to clathrin-coated pits in MCF-7 cells. Активация рецепторних тирозин киназ соответствующими лигандами приводит не только к распространению сигнала, но и инициирует разные процессы, например, клатрин-опосредованный эндоцитоз, которые регулируют клеточный ответ. Эти процессы строго контролируются благодаря координированному действию большого количества белков – ферментов, адапторов и ингибиторов. Примером адапторного белка, функционирующего как в эндоцитозе, так и в клеточной сигнализации, служит интерсектин 1 (ITSN1). Ранее нами показано, что ITSN1 формирует комплекс с адапторным белком Ruk/CIN85 и убиквитин лигазой Cbl-b, участвующими в негативной регуляции рецепторных тирозинкиназ. Целью данной работы было выявление субклеточной локализации комплексов Ruk/CIN85-ITSN1 по отношению к клатрину и Cbl-b. Методы. Для определения локализации белков клетки аденокарциномы молочной железы человека MCF-7 транзиентно трансфицировали конструкциями, кодирующими ITSN1 и клатрин, и исследовали методами прямого и не прямого иммунофлуоресцентного анализа. Результаты. Показано, что ITSN1 и Ruk/CIN85 частично колокализуются с Cbl-b и клатрином в клетках линии MCF-7. Выводы. Установлено, что комплексы ITSN1 и белка Ruk/CIN85 частично колокализуются с Cbl-b и находятся в клатрин-окаймленных ямках клеток линии MCF-7 Активація рецепторних тирозинкіназ відповідними лігандами призводить не лише до розповсюдження сигналу, але й ініціює різні процеси, серед яких клатрин-опосередкований ендоцитоз, що регулюють клітинну відповідь. Такі процеси чітко контролюються завдяки координованій дії великої кількості білків ферментів, адапторів та інгібіторів. Прикладом адапторного білка, що функціонує як в ендоцитозі, так і в клітинній сигналізації, є інтерсектин 1 (ITSN1). У попередніх дослідженнях ми показали, що ITSN1 утворює комплекс з адапторним білком Ruk/CIN85 та убіквітинлігазою Cbl-b, які залучені до негативної регуляції рецепторних тирозинкіназ. Метою даної роботи було визначи ти субклітинну локалізацію комплексів Ruk/CIN85-ITSN1 по відношенню до клатрину та Cbl-b. Методи. Для встановлення локалізації білків клітини аденокарциноми молочної залози людини MCF-7 транзієнтно трансфікували конструкціями, що містять ITSN1 і клатрин, та вивчали методами прямого і не прямого імунофлуоресцентного аналізу. Результати. Показано, що ITSN1 і Ruk/CIN85 частковоко локалізуються з Cbl-b і клатрином у клітинах лінії MCF-7. Висновки. Продемонстровано, що комплекси Ruk/ CIN85 з ITSN1 колокалізуються з Cbl-b і знаходяться у клатрин-облямованих ямках клітин лінії MCF-7. 2009 Article ITSN1 and Ruk/CIN85 colocalized to clathrin-coated pits in MCF-7 cells / O.V. Nikolaienko, I.Ya. Skrypkina, L.O. Tsyba, L.B. Drobot, A.V. Rynditch // Вiopolymers and Cell. — 2009. — Т. 25, № 5. — С. 424-427. — Бібліогр.: 16 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.0007F4 https://nasplib.isofts.kiev.ua/handle/123456789/152964 577.25 en Вiopolymers and Cell application/pdf Інститут молекулярної біології і генетики НАН України |
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Короткі повідомлення Короткі повідомлення |
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Короткі повідомлення Короткі повідомлення Nikolaienko, O.V. Skrypkina, I.Ya. Tsyba, L.O. Drobot, L.B. Rynditch, A.V. ITSN1 and Ruk/CIN85 colocalized to clathrin-coated pits in MCF-7 cells Вiopolymers and Cell |
| description |
Activation of receptor tyrosine kinases (RTK) by corresponding ligands results not only in signal propagation, but also initiates a number of processes, such as clathrin-mediated endocytosis, which precisely regulate biological outcome. These processes are tightly controlled by coordinated action of a plethora of proteins – enzymes, scaffolds and inhibitory molecules. An example of an endocytic accessory protein that also functions in cell signaling is provided by intersectin 1 (ITSN1). Previously we have shown that ITSN1 forms a complex with adaptor protein Ruk/CIN85 and ubiquitin ligase Cbl-b, which are implicated in down regulation of RTK. The present study aimed to determine the subcellular localization of ITSN1-Ruk/CIN85 complexes relatively to clathrin light chain and Cbl-b. Methods. Transient transfection of MCF-7 breast adenocarcinoma cells with the constructs containing Omni-tagged intersectin 1 and clathrin light chain fused with mCherry fluorescent protein was utilized to determine subcellular localization by direct or indirect immunofluorescence. Results. We found that Ruk/CIN85-ITSN1 complexes partially colocalized with Cbl-b and clathrin light chain in MCF-7 cells. Conclusions. In our report we provide experimental evidence that ITSN1-Ruk/CIN85 complexes exist in pre-assembled state with Cbl-b and are targeted to clathrin-coated pits in MCF-7 cells. |
| format |
Article |
| author |
Nikolaienko, O.V. Skrypkina, I.Ya. Tsyba, L.O. Drobot, L.B. Rynditch, A.V. |
| author_facet |
Nikolaienko, O.V. Skrypkina, I.Ya. Tsyba, L.O. Drobot, L.B. Rynditch, A.V. |
| author_sort |
Nikolaienko, O.V. |
| title |
ITSN1 and Ruk/CIN85 colocalized to clathrin-coated pits in MCF-7 cells |
| title_short |
ITSN1 and Ruk/CIN85 colocalized to clathrin-coated pits in MCF-7 cells |
| title_full |
ITSN1 and Ruk/CIN85 colocalized to clathrin-coated pits in MCF-7 cells |
| title_fullStr |
ITSN1 and Ruk/CIN85 colocalized to clathrin-coated pits in MCF-7 cells |
| title_full_unstemmed |
ITSN1 and Ruk/CIN85 colocalized to clathrin-coated pits in MCF-7 cells |
| title_sort |
itsn1 and ruk/cin85 colocalized to clathrin-coated pits in mcf-7 cells |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| publishDate |
2009 |
| topic_facet |
Короткі повідомлення |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/152964 |
| citation_txt |
ITSN1 and Ruk/CIN85 colocalized to clathrin-coated pits in MCF-7 cells / O.V. Nikolaienko, I.Ya. Skrypkina, L.O. Tsyba, L.B. Drobot, A.V. Rynditch // Вiopolymers and Cell. — 2009. — Т. 25, № 5. — С. 424-427. — Бібліогр.: 16 назв. — англ. |
| series |
Вiopolymers and Cell |
| work_keys_str_mv |
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| fulltext |
ÊÎÐÎÒʲ ÏβÄÎÌËÅÍÍß
ITSN1 and Ruk/CIN85 colocalized to clathrin-coated
pits in MCF-7 cells
O. V. Nikolaienko, I. Ya. Skrypkina, L. O. Tsyba, L. B. Drobot1, A. V. Rynditch
Institute of Molecular Biology and Genetics NAS of Ukraine
150, Zabolotnogo Str., Kyiv, Ukraine, 03680
1Palladin Institute of Biochemistry NAS of Ukraine
9, Leontovycha Str., Kyiv, Ukraine, 01601
rynditch@imbg.org.ua
Aim. Activation of receptor tyrosine kinases (RTK) by corresponding ligands results not only in signal
propagation, but also initiates a number of processes, such as clathrin-mediated endocytosis, which
precisely regulate biological outcome. These processes are tightly controlled by coordinated action of a
plethora of proteins – enzymes, scaffolds and inhibitory molecules. An example of an endocytic accessory
protein that also functions in cell signaling is provided by intersectin 1 (ITSN1). Previously we have shown
that ITSN1 forms a complex with adaptor protein Ruk/CIN85 and ubiquitin ligase Cbl-b, which are
implicated in down regulation of RTK. The present study aimed to determine the subcellular localization of
ITSN1-Ruk/CIN85 complexes relatively to clathrin light chain and Cbl-b. Methods. Transient transfection
of MCF-7 breast adenocarcinoma cells with the constructs containing Omni-tagged intersectin 1 and
clathrin light chain fused with mCherry fluorescent protein was utilized to determine subcellular
localization by direct or indirect immunofluorescence. Results. We found that Ruk/CIN85-ITSN1 complexes
partially colocalized with Cbl-b and clathrin light chain in MCF-7 cells. Conclusions. In our report we
provide experimental evidence that ITSN1-Ruk/CIN85 complexes exist in pre-assembled state with Cbl-b
and are targeted to clathrin-coated pits in MCF-7 cells.
Keywords: intersectin, Ruk/CIN85, Cbl-b, clathrin-coated pits, immunofluorescence.
Introduction. Intersectin 1 (ITSN1) is an evolutionary
conserved adaptor protein involved in the regulation of
multiple cellular processes [1–4]. Its ubiquitous
isoform comprises two N-terminal Eps15 homology
domains (EH1 and EH2), a central coiled-coil region
(CCR) and five tandem Src homology domains (SH3
A–E). By these domains ITSN1 was shown to bind
different endocytic proteins such as dynamin, epsin,
Eps15, synaptojanin 1, etc. (for review, see [5]). As any
other endocytic scaffolds, ITSN1 is also tightly linked
to both signal propagation and its attenuation. Mo-
reover, it is known that ITSN1 can directly interact
with MAPK cascade member Sos1 and ubiquitin
protein ligases c-Cbl and Cbl-b [6–8]. Recently, we
have found that ITSN1 forms a complex with another
signal attenuator, adaptor protein Ruk/CIN85 [5].
Furthermore, we have shown that Ruk/CIN85, c-Cbl
and ITSN1 colocalize in small (d = ~ 0.2–0.3 mm)
structures close to plasma membrane. In this report, we
describe localization pattern of Cbl-b and ITSN1-
Ruk/CIN85 complexes in MCF-7 cells and provide
evidence that ITSN1-Ruk/CIN85-rich structures
contain clathrin, and thus they may correspond to clath-
rin-coated pits (CCPs).
424
ISSN 0233-7657. Biopolymers and Cell. 2009. Ò. 25. N 5
Ó Institute of Molecular Biology and Genetics NAS of Ukraine, 2009
Materials and Methods. Antibodies. A
monoclonal anti-Omni (D-8): sc-7270, polyclonal
anti-Omni (M-21): sc-499 and polyclonal anti-Cbl-b
(H-454): sc-1705 antibodies were purchased from
Santa Cruz Biotechnology. For immunofluorescence,
Alexa Fluor 405 goat anti-rabbit IgG (Invitrogen) and
Texas Red horse anti-mouse IgG (Vector Laboratories
Inc.) antibodies were used.
Plasmid construction. The Omni epitope-tagged
ITSN1 construct was described previously [5]. Plasmid
containing mCherry-tagged clathrin light chain
(pmCherry-CLC) was constructed using vector
pEGFP-CLC kindly provided by Dr. Havrylov,
Poland.
Cell culture and transfection. The human breast
adenocarcinoma (MCF-7) cells stably overexpressing
GFP-fused Ruk/CIN85 (Drobot L., manuscript in
preparation) were grown on coverslips at 37 îÑ in 5 %
CO2 in Dulbecco’s modified Eagle’s medium with high
glucose content, 10 % fetal bovine serum, 2 mM
glutamine, 50 U/ml penicillin and 100 mg/ml strep-
tomycin. Cells were transfected using polyethylene-
imine (JetPEI, Polyplus Transfection) and processed
18 h after transfection.
Immunofluorescence and confocal microscopy.
Transfected cells were washed in cold PBS, fixed in
4 % formaldehyde for 15 min, washed three times for
5 min each with PBS containing 0.2 % Triton X-100
and blocked with blocking buffer (2 % bovine serum
albumine, 0.2 % Triton X-100 in PBS) for 30 min at
room temperature. Incubations were performed at
room temperature with antibodies diluted in blocking
buffer. Slides were mounted using PVA-DABCO
(Fluka) and images were captured with a Leica TSC
SPE or Zeiss LSM 510 Meta confocal microscopes. All
images represent a single confocal section, taken from
the bottom surface of expressing cells.
Results and Discussion. Clathrin-mediated
endocytosis (CME) is the major mechanism of protein
internalization in eukaryotic cell. While endocytosis
was traditionally considered as a negative regulatory
mechanism for activated RTKs, it is now clear that
positive signalling requires endocytosis as well.
During CME, receptors and their ligands are recruited
into clathrin-coated vesicles (CCVs) that are formed at
the plasma membrane in active sites called clathrin-
coated pits (CCPs). This process is facilitated by
adaptor proteins which interact directly or indirectly
with both cytosolic domains of receptors and clathrin.
It was shown that intersectin 1 localizes to pe-
rinuclear compartment and clathrin-coated pits of en-
dothelial and neuronal cells [9, 10]. In turn, Ruk/CIN85
was found at COPI-coated vesicles in Golgi [11, 12].
To determine whether ITSN1 and Ruk/CIN85 localize
within CCPs we analyzed the subcellular distribution
of these proteins in cells relatively to clathrin light
chain (CLC) – the central component of CCPs and
CCVs. MCF-7 cells stably overexpressing full-length
GFP-tagged Ruk/CIN85 were transiently transfected
with expression constructs encoding Omni-ITSN1 and
mCherry-tagged clathrin light chain (mCherry-CLC).
Immunofluorescence analysis revealed that GFP-Ruk/
CIN85 and Omni-ITSN1 proteins colocalized and
concentrated in the perinuclear region and in round
juxtamembrane structures, which were also clathrin-
positive (Fig. 1, see inset). Therefore, intersectin 1 and
Ruk/CIN85 partially localize to clathrin-coated pits in
MCF-7 cells.
It should also be noted that phenotype of MCF-7
cells stably overexpressing full-length GFP-tagged
Ruk/CIN85 resembles that of brefeldin A-treated HeLa
cells [13]: considerable number of cells contain
Ruk/CIN85-positive prolonged membrane tubules
(data not shown). This observation points to possible
perturbations in the membrane trafficking and is
consistent with a significant role of Ruk/CIN85 in
Golgi-endoplasmic reticulum and intra-Golgi transport
[11, 12].
Additionally, we examined the subcellular
colocalization of ITSN1, Ruk/CIN85 and the ubiquitin
ligase Cbl-b in MCF-7 cells. Cbl-b is known to interact
with both ITSN1 and Ruk/CIN85 adaptor proteins:
ITSN1 binds Cbl-b by its SH3 A and E domains [7],
while Ruk/CIN85 interacts with an atypical
proline-rich motif at the distal end of Cbl-b through
SH3 domains [14, 15]. Immunofluorescent analysis
revealed that Omni-ITSN1, GFP-Ruk/CIN85 and
endogenous Cbl-b display moderate levels of coloca-
lization in MCF-7 cells (Fig. 2, see inset).
Both ITSN1 and Ruk/CIN85 aid in assembling
clathrin-coated vesicles through association with
numerous endocytic proteins [5] and are presumed to
425
ITSN1 AND RUK/CIN85 COLOCALIZED TO CLATHRIN-COATED PITS IN MCF-7 CELLS
link receptor endocytosis with cytoskeletal rearran-
gements [4, 16]. It is widely accepted that numerous
components of signal transduction cascades assemble
at clathrin coated pits and remain associated during
endocytosis. In our report we provide additional
experimental evidence that supports this idea. How-
ever, determination of the exact composition of
complexes and elucidation of their localization-critical
components require additional experiments.
Î. Â. ͳêîëàºíêî, ². ß. Ñêðèïê³íà, Ë. Î. Öèáà, Ë. Á. Äðî áîò,
À. Â. Ðèí äè÷
²íòåð ñåê òèí 1 òà Ruk/CIN85 êî ëî êàë³çó þòü ñÿ â êëàò ðèí-
îá ëÿ ìî âà íèõ ÿì êàõ êë³òèí ë³í³¿ MCF-7
Ðå çþ ìå
Ìåòà. Àêòèâàö³ÿ ðå öåï òîð íèõ òè ðî çèíê³íàç â³äïîâ³äíè ìè
ë³ãàí äà ìè ïðè çâî äèòü íå ëèøå äî ðîç ïîâ ñþä æåí íÿ ñèã íà ëó, àëå
é ³í³ö³þº ð³çí³ ïðî öå ñè, ñå ðåä ÿêèõ êëàò ðèí-îïî ñå ðåä êî âà íèé
åí äî öè òîç, ùî ðå ãó ëþ þòü êë³òèí íó â³äïîâ³äü. Òàê³ ïðî öå ñè
÷³òêî êîí òðî ëþ þòü ñÿ çà âäÿ êè êî îð äè íî âàí³é 䳿 âå ëè êî¿
ê³ëüêîñò³ á³ëê³â – ôåð ìåíò³â, àäàï òîð³â òà ³íã³á³òîð³â. Ïðèê -
ëà äîì àäàï òîð íî ãî á³ëêà, ùî ôóíêö³îíóº ÿê â åí äî öè òîç³, òàê ³
â êë³òèíí³é ñèã íàë³çàö³¿, º ³íòåð ñåê òèí 1 (ITSN1). Ó ïî ïå ðåäí³õ
äîñë³äæåí íÿõ ìè ïî êà çà ëè, ùî ITSN1 óòâî ðþº êîì ïëåêñ ç àäàï -
òîð íèì á³ëêîì Ruk/CIN85 òà óá³êâ³òèíë³ãà çîþ Cbl-b, ÿê³ çà ëó -
÷åí³ äî íå ãà òèâ íî¿ ðå ãó ëÿö³¿ ðå öåï òîð íèõ òè ðî çèíê³íàç. Ìå-
òîþ äà íî¿ ðî áî òè áóëî âèç íà ÷è òè ñóá êë³òèí íó ëî êàë³çàö³þ
êîì ïëåêñ³â Ruk/CIN85-ITSN1 ïî â³äíî øåí íþ äî êëàò ðè íó òà
Cbl-b. Ìå òî äè. Äëÿ âñòà íîâ ëåí íÿ ëî êàë³çàö³¿ á³ëê³â êë³òèíè
àäå íî êàð öè íî ìè ìî ëî÷ íî¿ çà ëî çè ëþ äè íè MCF-7 òðàí糺íòíî
òðàíñô³êó âà ëè êî íñòðóêö³ÿìè, ùî ì³ñòÿòü ITSN1 ³ êëàò ðèí,
òà âèâ ÷à ëè ìå òî äà ìè ïðÿ ìî ãî ³ íå ïðÿ ìî ãî ³ìó íîô ëó î ðåñ öåí -
òíî ãî àíàë³çó. Ðå çóëü òà òè. Ïî êà çà íî, ùî ITSN1 ³ Ruk/CIN85
÷àñ òêî âî êî ëî êàë³çó þòü ñÿ ç Cbl-b ³ êëàò ðè íîì ó êë³òè íàõ ë³í³¿
MCF-7. Âèñ íîâ êè. Ïðî äå ìî íñòðî âà íî, ùî êîì ïëåê ñè Ruk/
CIN85 ç ITSN1 êî ëî êàë³çó þòü ñÿ ç Cbl-b ³ çíà õî äÿòü ñÿ ó êëàò -
ðèí-îá ëÿ ìî âà íèõ ÿì êàõ êë³òèí ë³í³¿ MCF-7.
Êëþ ÷îâ³ ñëî âà: ³íòåð ñåê òèí, Ruk/CIN85, Cbl-b, êëàò ðèí-îá -
ëÿ ìî âàí³ ÿìêè, ³ìó íîô ëó î ðåñ öåíö³ÿ.
À. Â. Íè êî ëà åí êî, È. ß. Ñêðèï êè íà, Ë. À. Öèáà, Ë. Á. Äðî áîò,
À. Â. Ðûí äè÷
Èíòåð ñåê òèí 1 è Ruk/CIN85 êî ëî êà ëè çó þò ñÿ â êëàò ðèí-
îêàé ìëåí íûõ ÿì êàõ êëå òîê ëè íèè MCF-7
Ðå çþ ìå
Öåëü. Àêòèâàöèÿ ðå öåï òîð íèõ òè ðî çèí êè íàç ñî îò âå òñòâó þ -
ùè ìè ëè ãàí äà ìè ïðè âî äèò íå òîëü êî ê ðàñ ïðîñ òðà íå íèþ ñèã -
íà ëà, íî è èíè öè è ðó åò ðàç íûå ïðî öåñ ñû, íà ïðè ìåð, êëàò ðèí-
îïîñ ðå äî âàí íûé ýí äî öè òîç, êî òî ðûå ðå ãó ëè ðó þò êëå òî÷ íûé
îò âåò. Ýòè ïðî öåñ ñû ñòðî ãî êîí òðî ëè ðó þò ñÿ áëà ãî äà ðÿ êî -
îð äè íè ðî âàí íî ìó äå éñòâèþ áîëü øî ãî êî ëè ÷åñ òâà áåë êîâ –
ôåð ìåí òîâ, àäàï òî ðîâ è èí ãè áè òî ðîâ. Ïðè ìå ðîì àäàï òîð íî -
ãî áåë êà, ôóíê öè î íè ðó þ ùå ãî êàê â ýí äî öè òî çå, òàê è â êëå òî÷ -
íîé ñèã íà ëè çà öèè, ñëó æèò èí òåð ñåê òèí 1 (ITSN1). Ðà íåå íàìè
ïî êà çà íî, ÷òî ITSN1 ôîð ìè ðó åò êîì ïëåêñ ñ àäàï òîð íûì áåë -
êîì Ruk/CIN85 è óáèê âè òèí ëè ãà çîé Cbl-b, ó÷àñ òâó þ ùè ìè â íå -
ãà òèâ íîé ðå ãó ëÿ öèè ðå öåï òîð íûõ òè ðî çèí êè íàç. Öåëüþ äàí íîé
ðà áî òû áûëî âû ÿâ ëå íèå ñóá êëå òî÷ íîé ëî êà ëè çà öèè êîì ïëåê ñîâ
Ruk/CIN85-ITSN1 ïî îò íî øå íèþ ê êëàò ðè íó è Cbl-b. Ìå òî äû.
Äëÿ îïðå äå ëå íèÿ ëî êà ëè çà öèè áåë êîâ êëåò êè àäå íî êàð öè íî ìû
ìî ëî÷ íîé æå ëå çû ÷å ëî âå êà MCF-7 òðàí çè åí òíî òðàíñ ôè öè ðî -
âà ëè êî íñòðóê öè ÿ ìè, êî äè ðó þ ùè ìè ITSN1 è êëàò ðèí, è èñ ñëå -
äî âàëè ìå òî äà ìè ïðÿ ìî ãî è íå ïðÿ ìî ãî èì ìó íîô ëó î ðåñ-
öåí òíî ãî àíà ëè çà. Ðå çóëü òà òû. Ïî êà çà íî, ÷òî ITSN1 è Ruk/
CIN85 ÷àñ òè÷ íî êî ëî êà ëè çó þò ñÿ ñ Cbl-b è êëàò ðè íîì â êëåò -
êàõ ëè íèè MCF-7. Âû âî äû. Óñòà íîâ ëå íî, ÷òî êîì ïëåê ñû ITSN1
è áåë êà Ruk/CIN85 ÷àñ òè÷ íî êî ëî êà ëè çó þò ñÿ ñ Cbl-b è íà õî -
äÿò ñÿ â êëàò ðèí-îêàé ìëåí íûõ ÿì êàõ êëåòîê ëèíèè MCF-7.
Êëþ ÷å âûå ñëî âà: èí òåð ñåê òèí, Ruk/CIN85, Cbl-b, êëàò ðèí-
îêàé ìëåí íûå ÿìêè, èì ìó íîô ëó î ðåñ öåí öèÿ.
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Íàä³éøëà äî ðå äàêö³¿ 10.07.09
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ITSN1 AND RUK/CIN85 COLOCALIZED TO CLATHRIN-COATED PITS IN MCF-7 CELLS
Ðè ñóí êè äî ñòàòò³ O. Â. ͳêî ëàºíêî òà ñï³âàâò.
ISSN 0233-7657. Biopolymers and Cell. 2009. Ò. 25. ¹ 5
Fig. 1. ITSN1 colocalizes with Ruk/CIN85 and clathrin light chain in MCF-7 cells. MCF-7 cells stably overexpressing GFP-tagged
Ruk/CIN85 were transfected with Omni-ITSN1-s and mCherry-CLC. ITSN1 protein was localized by indirect immunofluorescence with
anti-Omni antibodies followed by secondary antibodies coupled to Alexa Fluor 405 (blue). White arrows indicate some of structures
labelled by all three proteins
Fig. 2. ITSN1 colocalizes with Ruk/CIN85 and Cbl-b in MCF-7 cells. MCF-7 cells stably overexpressing GFP-tagged Ruk/CIN85 were
transfected with Omni-ITSN1-s and double-stained with antibodies against Omni-tag (visualized with Texas Red-conjugated anti-mouse
IgG) and endogenous Cbl-b (visualized with Alexa Fluor 405-conjugated anti-rabbit IgG). White arrows indicate some of structures that are
positive for all three proteins. Blunt white arrows indicate structures negative for at least one of the proteins
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