Download Cystic Fibrosis

Cystic Fibrosis
Cell Biology of mutations in Chloride
channels and their defective transport
Michael Lohmüller
http://dokhlab.unc.edu/research/CFTR/images/CFTR-model.jpg
Cystic Fibrosis
•  autosomal recessive
•  loss of function of CFTR
•  Multisystem disorder
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http://en academic r
http://en.academic.ru/pictures/enwiki/67/Cystic_fibrosis_manifestations.png
CFTR
•  Cystic Fibrosis transmembrane conductance
regulator
•  transmembrane protein
•  chloride channel
•  mutations lead to
alteration in fluid transport
•  expressed in the apical
membrane of epithel cells
https://upload.wikimedia.org/wikipedia/commons/thumb/0/03/
Protein_CFTR_PDB_1xmi.png/800px-Protein_CFTR_PDB_1xmi.png
Physiological localization
mouse intestine
Ameen et al. 2012
Pathophysiology of the lungs
http://learn.genetics.utah.edu/content/disorders/singlegene/cf/images/cf-channel.jpg
http://www.nhlbi.nih.gov/sites/www.nhlbi.nih.gov/files/images_252
CFTR: Deficient in Localization / Function
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Drug Treatment
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Experiment 1
Generation of Cell lines: (by I. Vietor)
HeLa + CFTR WT-GFP
HeLa + CFTR ΔF508-GFP
? Localization of CFTR and of CFTR ΔF508 in the
HeLa cell line?
Experiment 1
Generation of Cell lines: (by I. Vietor)
HeLa + CFTR WT-GFP
HeLa + CFTR ΔF508-GFP
cultivate on cover slips
fixation with 4%PFA
Staining anti-Protein-Disulphide-Isomerase (PDI)
Counterstaining HOECHST
Confocal microscopy
HeLa – CFTR WT
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DNA
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DNA
CFTR-GFP
PDI
CFTR-GFP
PDI
HeLa – CFTR F508
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DNA
CFTR-GFP
PDI
CFTR-GFP
PDI
Localization of CFTR / CFTR ΔF508
•  CFTR-WT expressed in cytoplasm
•  CFTR F508 expressed
CFTRWT
–  focal
–  lower concentration
–  perinuclear
CFTR
F508
http://www.frontiersin.org/files/Articles/32448/fphar-03-00160-HTML/image_m/
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Glycosylation Profile
Edelmann et al. 2015
Drug Treatment
•  CFTR corrector
–  folding and trafficking
•  potentiator
–  enhancing the open probaility
•  proteasome inhibitor
•  low temperature
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Experiment 2
Generation of Cell lines: (by I. Vietor)
HeLa Cells (-CFTR)
HeLa + CFTR WT-GFP
HeLa + CFTR ΔF508-GFP
Glycolyzation profile of CFTR WT and
ΔF508?
CFTRΔ
Glycolyzation profile after treatment?
Experiment 2
Generation of Cell lines: (by I. Vietor)
HeLa Cells (-CFTR)
HeLa + CFTR WT-GFP
HeLa + CFTR ΔF508-GFP
cultivation
20h
treatment
cell lysis
ΔF508 + ALLN (20μM) (PI)
ΔF508 + VX809 (5μM) (Corrector)
ΔF508 + VX809(30μM)(Corrector)
ΔF508 + VX770(5μM) (Potentiator)
ΔF508 + VX770(30μM) (Potentiator)
ΔF508 @ 27C
SDS-Page-Gel/
Western Blot
ALLN - PI
VX809 - Corrector
VX770 - Potentiator
WB of HeLa cells after 20h Drug
Treatment
Future Experiments:
-Combination of treatment
-lowering the treatment duration
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Vij et al. 2010
Experiment 3
Generation of Cell lines: (by I. Vietor)
Hek293 Cells
Hek293 Cells + CFTR WT
Hek293 Cells + CFTR ΔF508
CFTR regulated NF-kB activity?
Experiment 3
treatment
LUCIFERASE MEASUREMENT
FL/RL
FL/RL
+TNF
FL/RL
FL/RL
+TNF
CFTR regulated NF-kB activity?
REPETITION
THANK YOU FOR YOUR ATTENTION
Figure 7. Fluorescence microcopy of yeast strains analysing the MVB
and autophagy functionality.
Results
Figure 6. Trafficking scheme of CPS and Atg8 in the MVB and
autophagy pathway.
Figure 5. Experimental setup of MVB and autophagy analysis.
MVB/AUTOPHAGY FUNCTION ANALYSIS
Figure 1. Functions of CORVET and HOPS within the
endolysosomal pathway. (Ungermann et al. 2013)
Michael Lohmüller
Phenotype of ARC syndrome patient mutations in
yeast and their role in the MVB and autophagy
pathway
ABSTRACT
The Arthrogryposis Renal Dysfunction Cholestasis Syndrome, ARC Syndrome,
is an autosomal recessive disease with variable severity. Defects of the
VPS33B lead to a multisystem disorder. The VPS33 protein is a vasculor
protein sorting protein with two isoformes in human. VPS33 is involved in two
different multiprotein complexes, CORVET and HOPS. These tethering
complexes bring membranes in close proximity before fusion and are involved
in maturation of late endosomes and MVB, formation of vacuoles and
lysosome, the AP3 pathway and the transport from Golgi complex to the early
endosomes. The mutations of ARC Syndrome patients have a loss of function
of the VPS33 and therefore alterations of the CORVET and HOPS complex.
The aim of our study was to introduce three mutations from ARC-syndrome
patients in the yeast homolog of VPS33 and analyze the phenotype and the
influences on the MVB and autophagy pathway.
CLONING ANALYSIS OF ARC PATIENT MUTATIONS
Figure 2. Experimental setup of cloning analysis of patient mutations.
Results
Figure 3. Growing
phenotype of transformed yeast strains
on YNB-Leu and
YNB-Leu + 0.6µg/ml
Canavanin plates for
72h at 26°C.
Figure 4. Live cell microscopy of transformed yeast strains.
Summary
www.postersession.com
•  The patient mutations A483* and L22P display reduced growth on canavanin medium (Figure 3).
•  A483* and L22P display many small dots instead of one large vacuole in the FM4-64 staining (Figure 4.), similar to the vps33Δ.
•  T291F grows like wild type on canavanin and also display same vacuole (Figure 3 and 4).
•  VPS33Δ strains are deficient in the MVB and in the autophagosomal pathway (Figure 7.) and cannot form intact vacuoles. (Figure 4.).
M.E. Lohmüller1, S. Avci1, S.H.J. Heeke1, U. Kühbacher1, H.G. Wurzer1,
M.Heß2, M.E. de Araujo3, T. Stasyk3 and L. A. Huber3
1
Tuberous sclerosis complex is a rare autosomal dominant disease. This crucial multi
system disease can nearly affect every organ
system and cause benign tumors in brain,
kidneys, heart and other organs. The leading
cause for these symptoms is a mutation in the
tuberous sclerosis complex (TSC) 1 or 2 which
inhibits the mTORC1 (mammalian target of
rapamycin complex 1). The mutations in TSC1
and 2 lead to a hyperactivation of the protein
kinase mTORC1 which nearly affects every
cellular metabolism process. These processes
are regulated physiologically by growth factors
like insulin or amino acid concentrations in the
lysosome. Insulin mediates through the IGF
and thePI3K pathway. This pathway inhibits by
insulin signaling the phosphorylation of AKT
and therefore the inhibition of TSC. TSC in it’s
active state acts as GAP (GTPase activating
proteins) of Rheb which activates mTORC1
bound to GFP. The amino acid state is sensed
by LAMTOR in the inner lumen of lysosomes.
and activates mTORC1(Manning et al. 2014).
The active mTORC1 can activate through the
kinase activity several kinases or directly
phosphorylates proteins and transcription
Introduction
TSC2KO cells in the amount of their
organelles, especially the
amount of
mitochondria increased vastly.
1 Student Molecular Medicine Master, 2 Division of Histology and Embryology and Division of Cell
Biology, 3 Innsbruck Medical University, 6020 Innsbruck, Austria
January 2016.
Abstract
Tuberous sclerosis complex is a severe
multi system disorder which is identified by
a distinct pathology, called hamartomas
(Gomez et al. 1988). These benign cell
lesions are found in nearly all organs and
can lead to seizures, pathology of the
organs, and other phenotypes which can
vary in patients. The mammalian target of
rapamycin (mTOR) is a major regulator of
cellular processes e.g. cell proliferation,
cell growth, translation, autophagy and
mitochondria biogenesis. The mTOR
complex is inhibited by the tumor
suppressor TSC1 and TSC2 which are
mutated in the TSC patients. The macrolide
rapamycin produced by bacteria can inhibit
the mTOR complex, but inhibits just
selectively in some patients and not in all
(Sehgal et al. 1975). Therefore the
metabolites of the mTOR pathway have to
be further analyzed to find potential drug
treatment options for the individual
tuberous sclerosis patients. In our study
we analyzed in TSC2 KO HeLa cells the
proteins in the mTORC1 signaling pathway
under different treatment options. The S6
protein, subunit of the ribosomal protein, is
activated straightforward, in contrast to
ULK1, autophagy activator, which is
inhibited under more complex conditions.
Further we found tremendous changes in
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factor and therefore regulates the cellular
metabolism. ULK-1 a protein of the autophagy
pathway is phosphorylated during mTORC1
activity and therefore autophagy is inhibited.
Autophagy and lysosomal proteins are also
regulated on transcriptional level by the
transcription factor EB (TFEB) (Ferguson et al.
2012). TFEB is phosphorylated by the mTOR
complex and doesn’t translocate to the
nucleus where it acts as transcription factor.
Phosphorylation also activate cellular
processes, e.g. the phosphorylation of the S6
ribosomal subunit which activates translation.
In the case of hyperactivation of mTORC1,
due to the missing suppression of TSC, there
is a imbalance of cellular processes which lead
to benign phenotype of cells.
The discovery of the mechanisms of sirolimus,
also known as rapamycin, (Sehgal et al. 1975)
which is a specific inhibitor of mTORC1
contributed to the field of tuberous sclerosis
complex as a drug treatment. The treatments
with sirolimus display a great diversity in
patients (Katulska et al.2012). Seizure patients
vary from no seizures to no effect on
rapamycin. The individual pathology of
patients with mutations in one pathway,
respectively one protein, display the
complexity of this multi system disorder.
We investigated the up- and downstream
protein expressions of mTORC1 pathway
involved proteins, in TSC deficient cell lines,
during starvation, insulin stimulation and
rapamycin treatment. Further we analyzed the
translocation of TFEB in the cell lines during
treatments, starvation and stimulation. Cellular
phenotype of cell lines were observed in the
transmission electron microscope and also
LAMTOR2(p14) KO mouse embryonic fibroblasts (MEFs) were generated to analyze the
influence on cellular morphology. The aim of
our studies was to shed new light on the
pathways involved in the TSC2KO pathology
to generate new ideas for treatment options for
previously untreated patients.
Results
Signaling aberrations of mTORC1
during starvation, stimulation and drug
treatment
The influence of insulin, amino acid starvation
and rapamycin on mTORC1 signaling were
already described previously (Manning et al.
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2014) (Fig. 1A). We used combinations of
treatments in cell culture experiments,
analyzed via SDS-PAGE and Western blotting
to address the question whether the up- and
downstream targets are influenced by the
treatments and their combinations.
The results (Fig. 1B) confirmed the generation
of the TSC2 KO cell line, despite there is still
signal in the KO for TSC2.
Total AKT, which partially serves as loading
control, displays similar signals in all probes,
whereas the phosphorylation and therefore
activation of AKT was induced in the cells
treated with insulin, stronger with insulin or
FBS with rapamycin in the WT cells. The
TSC2KO displays similar P-AKT signals with
the FBS or insulin with rapamycin, which
verified the described feedback mechanisms
of inactive mTORC1 towards AKT (Hsu et al.
2011).
The phosphorylation of ULK1 as inhibition of
autophagy was induced in the TSC2 KO cells
without rapamycin. The WT HeLa cells and the
TSC2 KO cells displayed basal signal for
ULK1 phosphorylation and therefore perform
autophagy.
In contrast to the phosphorylation of ULK1, the
phosphorylation of S6 leads to active
translation due to mTORC1 activity. Starved,
untreated and unstimulated cells doesn’t
exhibit S6 phosphorylation, neither in the WT
nor the TSC2 KO cells. Merely insulin or
insulin with rapamycin display bands, in
contrast to that the FBS+ treatment and FBS+
with rapamycin display a decreased signal.
The treatments of insulin and rapamycin look
similar in TSC2KO cells, but the FBS+ and
FBS+ with rapamycin treated cells show
increased bands compared to the WT.
Total S6 bands, display similar intensities with
all treatments, however the TSC2KO cells
exhibit a small increase in intensity.
TFEB localization dependent on TSC2
during starvation, stimulation and drug
treatment
Transcription factor EB (TFEB) is a major
transcription factor and regulator for lysosomal
and autophagy biogenesis. Therefore we
starved, stimulated and treated HeLa WT cells
and TSC2KO cells with torin1 and analyzed
the localization pattern of TFEB (Fig. 2C).
Physiological TFEB is localized partially in the
cytosol and the nucleus (Fig. 2A). Starvation or
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TSC2KO
WT
FBS
B
Rapamycin (40µM)
Insulin (100nM)
TSC2 (200 KDa)
P-ULK1 (140 KDa)
3
total-AKT (60 KDa)
P-AKT (62 KDa)
total S6 (35 KDa)
P-S6 (35 KDa)
Figure 1. Insulin, serum starvation and rapamycin influence the up and downstream
pathway participants of mTORC1.
(A) Schematic of the AKT-TSC-mTORC1 -p14-pathway through wich insulin and amino acids influence the
mTORC1 activity. of
(B) HeLa cells and TSC2KO HeLa cells were starved (24 h) treated with rapamycin (24 h) and
subsequently stimulated with insulin (30 min). Cells were lysed and protein concentrations wee
determined with BCA protein assay. Protein lysates (30μg) were separated with a 12% SDS-PAGE gel
and transferred to a PVPF membrane for antibody incubation.
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Subcellular phenotype of steady state
and starved HeLa cells and TSC2 KO
cells
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LAMP1
LAMP1
TFEB
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LAMP1
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The mTORC1 signaling pathway influences
many different aspects of cell biology
metabolism. TSC2 is one major inhibitor of
mTORC1, therefore we analyzed the
subcellular phenotypes with transmission
electron microscopy during steady state and
starvation.
HeLa WT cells under steady state conditions
and starvation displayed several endosomes,
lysosomes and probably multi-vesicular-bodies
(Fig. 3AB). LAMP 1 signal increased relative
during starvation. In contrast to that decreased
LAMP1 signal and decreased amount of
endosomes was observed in the TSC2 KO
LAMP1
TFEB
starvation (80’)
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torin1 (3h)
TFEB
starvation (60’) + stimulation (20’)
TFEB
control
treatment with torin1 leads to a translocation of
TFEB to the nucleus and starvation with
subsequent stimulation directs TFEB back to
the cytosol (Fig. 2A).
TFEB is localized in TSC2 KO cells during
steady state in the cytoplasm. Starvation and
starvation with subsequent stimulation lead to
a partial translocation to nucleus. Torin1
treated TSC2 KO cells displayed in one image
perinuclear localization, despite in all other
slides TFEB translocated to the nucleus (Fig.
2B).
A
HeLa WT
HeLa WT
B
C
LAMP1
TFEB
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LAMP1
starvation (80’)
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control
TFEB
LAMP1
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starvation (60’) + stimulation (20’)
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(A) Immunofluorescence images of control, torin 1 treated, amino acids starved and, amino acids
starved and stimulated HeLa cells. HeLa cells express TFEB tagged with GFP after transfection.
Cells were stained for LAMP1 (Alexa-568, red) and nucleic acid (Hoechst 33342).
(B) TSC2KO HeLa cells were treated as in (A).
(C) Schematic of the TSC2-mTORC1-TFEB pathway through which amino acids and insulin influence
the gene expression of lysosomal and autophagic genes.
Figure 2.Amino acids starvation and torin1
treatment influence the translocation of TFEB
as a major regulator of the lysosomal and
autophagic gene expression.
TSC2 KO
TSC2 KO
TSC2 KO
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(mitochondria).
Figure 3. Subcellular phenotype of TSC2KO cells and WT cells under starvation
and steady state.
(A) TEM image of HeLa WT cells stained anti LAMP1 with silver enhancement.
(B) HeLa WT starved (24h) treated as in (A)
(C/E) TSC2KO steady state and (D/F) TSC2KO starved treated as in (A).
Lettering: n (nucleus), E (endosome), G (Golgi), MVB (multi vesicular body) and
*
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cells (Fig. 3CD). Lysosomes appear smaller
and intra luminal vesicles (ILV’s) of MVB
relative large in diameter. Differences in the
phenotype between the TSC2KO cells during
steady state and starvation was not analyzed.
Distinction in phenotype have to be analyzed
further with quantification, but a increase of
mitochondria was studied in the TSC2KO cells
in contrast to the WT cells (Fig. 3EF).
Cell diameter variation in LAMTOR2
deficient MEF cell lines.
The late endosomal adaptor molecule
(LAMTOR) is a major sensor for amino acids
in the mTOR pathway, which is associated to
the endosomal membrane and contributes to
the mTORC1 pathway due to the Rag
heterodimers. This multiprotein complex
consists of LAMTOR1 (p18), LAMTOR2 (p14),
LAMTOR3 (MP1), LAMTOR4 (HPXIP) and
LAMTOR5 (C7orf59). In this experiment we
analyzed the cell diameter, which is dependent
on the single allele KO of p14 (-/fl), double
allelic KO of p14 (-/-) and the reconstituted
p14-GFP in -/- p14 cells (rec).
The cell diameter increases in the -/- p14
MEFs (Fig. 4AB). The reconstitution decreases
the cell diameter, but not back to the normal
diameter of the -/fl cells (Fig.4C).
Discussion
The understanding of mTORC1 regulation
mechanisms and the proteins which are part of
the pathway is very important for the
development of drugs, e.g. tuberous sclerosis
complex or cancer patients. The phosphorylation status of AKT gives rise, that there
is a very important feedback regulation,
especially during inhibition of mTORC1 by
rapamycin, already described earlier (Hsu et
al. 2011). The exact mechanisms have to be
further studied, also as a possible drug target.
The inhibition of autophagy is just visible in the
TSC2 KO cells without inhibition of mTORC1.
This displays that autophagy is not a primary
pathway which is inhibited during mTORC1
activation. Autophagy is also needed in
mTORC1 active cells, due to the fact that the
nutrient supply can diversify over time.
S6 as part of the ribosomal S40 subunit is
important for the regulation of translation.
Phosphorylation leads to an activation of the
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translation machinery and is mediated by
active mTORC1 through insulin. FBS+ serum
as a induction leads to a reduced phosphorylation compared to the insulin
stimulation, which results in phosphorylation of
S6, although rapamycin inhibits mTORC1. The
reduction of activation under FBS+ treatment
is missing in the TSC2 KO cells. The general
expression of S6 shows a relative increase in
the TSC2 KO cells which could be further
analyzed in future experiments with qPCR.
The phosphorylation of S6 and the influence
on translation could be identified as a early
action of mTORC1, even under serum
starvation or inhibition with rapamycin.
Lysosomal and autophagic gene expression
induction was also described by translocation
of TFEB to the nucleus after mTORC1
inhibition was also described and this could be
also a potential target as treatment option, or
as read out for future experiments. The fact
that one of five specimens after torin1
treatment, doesn’t display a translocation of
TFEB to the nucleus could be due to variation
during the drug treatment, but will be further
analyzed in future experiments.
The subcellular analyzations of organelles in
the TEM clearly displayed that the LAMP1
staining is decreased in TSC2KO cells, which
could be a read out of the missing
translocation of TFEB. The reduced quality of
the images is based on the chemical fixation of
the specimens and could be enhanced for
quantitative analysis of endosomes and
lysosomes in future by application of cryo-EM.
The tremendous increase of mitochondria in
the TSC2 KO cells is obvious in the chemical
fixed specimens, but could be quantified in
future slides with cryo-EM. The influence of
active mTORC1 to mitochondria is
unambiguous displayed in the TSC2 KO cells
and will be further analyzed in future cell
culture experiments, to shed new light on the
unknown pathways which activate the
mitochondria biogenesis.
LAMTOR2 as part of the lysosomal amino acid
sensing machinery and mTORC1 activation,
also contributes to the cellular biogenesis,
which was shown with the read out of the
increased cell diameter in the p14 KO. The
fact that the influence couldn’t be rescued by
the reconstituted cells, could be based on the
GFP tag. LAMTOR (14KDa) could be
influenced by the relative large GFP (26.9KDa)
in it’s function. Therefore in future experiments
the p14 shouldn’t be GFP tagged, to prevent
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Figure 4. Cell diameter measurement of p14-/-; p14 fl/-; and p14-/- p14-GFP MEF cells
with Countess cell counter.
(A) Graph of counted cells with specific diameter.
(B) Box plots of cell diameter of p14 -/- ; p14fl/- and p14 rec MEFs.
(C) Table for viability, cell number and cell diameter of the -/-, fl/- and rec cells.
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influences on the function in the reconstituted
cells.
Our experiments confirmed the previous
findings for the up- an downstream targets of
mTORC1 and the alterations due to insulin,
FBS and rapamycin/torrin1 treatment. We
propose that the phosphorylation of S6 is an
straightforward action of mTORC1 activation in
contrast to the phosphorylation of ULK1 which
appears more regulated. Further we could
display in TEM that TSC2 KO lead to a
tremendous change in subcellular metabolism,
especially in mitochondria biogenesis. These
findings and the lysosome and endosome
expression have to be validated quantitative in
cryo-EM. LAMTOR2 as another contributor
towards the cellular phenotype could also be
identified in our studies.
Material and Methods
Cell Culture
HeLa WT and TSC2 KO HeLa cells were
cultivated in Dulbecco’s Modified Eagle’s
Medium (DMEM), 10%FBS and 1% Penstrep
at 37°C wit 5%CO2 and 95% humidity. Cells
were provided by the Division of Cell Biology
Innsbruck, Medical University.
For starvation and drug treatment experiment,
the cells were rinsed with pre-warmed FBSfree or complete medium and treated with
FBS-free medium, FBS-free medium with
40μM rapamycin, complete growth medium or
complete growth medium with 40μM
rapamcyin for 24h at 37°C. For stimulation
experiments the cells were stimulated after
starvation with or without Rapamycin for
30minutes with 100nM insulin in FBS-free
medium. Subsequently the cells were washed
with ice-cold PBS and lysed with 150μl of Cell
lysis buffer (50mM Tris-HCl pH 8.0, 150mM
NaCl, 1% Triton X-100, 10% glycerol, 1mM
EDTA, supplemented with protease inhibitors
(10μg/ml aprotinin, 10μg/ml leupeptin, 1μg/ml
pepstatin, 0.4mM Pefabloc) and phosphatase
inhibitors (50mM NaF, 5mM Na4P2O7 and 1mM
Na3OV4) on ice. The lysates were used for
SDS-PAGE and Western blotting.
Starvation for the immunofluorescence
experiment was performed with DMEM Ham’s
F-12 w/Hepes, NaHCO3, w/o aminoacids, 1%
Penstrep, pH 7.4 for 80 minutes at 37°C. The
stimulated cells were starved for 60 minutes at
37°C and then stimulated for 20minutes with
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DMEM, 10%FBS and 1% Penstrep at 37°C.
Drug treatment was achieved with DMEM,
10% FBS and 1% Penstrep supplemented with
330nM Torin1 for 3h at 37°C. The steady state
control was incubated with DMEM, 100% FBS
and 1% Penstrep for 3h at 37°C.
Transmission electron microscopy specimen
were prepared starved as the specimen for
immunofluorescence. Subsequent they were
fixed in 4% PFA for pre-embedding.
p14-/-, p14f/- and p14-/- GFPp14 MEFs were
cultivated with DMEM, 10%FBS and 1%
Penstrep for cell diameter experiments.
Cell line establishment
TSC2KO HeLa cells were generated with
CRISPR/CAS9 by the Division of Cell Biology,
Innsbruck. The protein product for p53 was
also HPV inactivated, to prevent early
senescence. TSC2 KO cells and HeLa WT
cells were transfected with GFP tagged TFEB.
p14-/-, p14f/- and p14-/- GFPp14 MEFs were
provided by the Division of Cell Biology,
Innsbruck, via Cre-Lox-recombination (Teis et
al. 2006).
SDS-PAGE and Western Blotting
The protein concentrations were determined
with a BCA protein determination.15μg protein
of each sample were adjusted with lysis buffer
and 4x SDS-sample buffer to the same
volume. The probes were boiled at 95°C for 5
minutes and centrifuged for 5 minutes with
13000g and subsequently loaded onto a 12%
SDS gel. The gel electrophoresis was runned
at 25mA per gel and set 125V as maximum for
90 minutes.
Subsequently the proteins were transferred
onto a PVPF membrane via western blotting at
125mA per 2 gels for 90 minutes.
The membranes were stained with amido
black for 1 minute and labelled.
The membranes were washed with washing
buffer, 50mM Tris-HCl pH 8.0, 100mM NaCl,
0.1% Tween 20, blocked with blocking buffer,
5% milk powder, and incubated with antibodies
in binding buffer, 3% BSA in washing buffer,
0.02% NaN3, over night.
The membranes were incubated with
secondary antibody in blocking buffer for 45
minutes at room temperature. The membranes
were finally incubated with ECL-substrate and
exposed to films in the dark room.
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Immunofluorescence analysis
HeLa cells and TSC2 KO cells were cultured
on cover slips. The cells were fixed with 4%
PFA solution, 4% PFA in Cytoskeletal buffer
(CB), 10mM Pipes pH6.8, 150mM NaCl, 5mM
EGTA, 5mM Glucose, 5mM MgCl2, for 8
minutes. Cells were washed with CB and
blocked with blocking solution, 2% gelatine,
50mM NH4Cl, 0.025% Saponin in CB, for 30
minutes. Primary and secondary antibody
were diluted in blocking buffer. Specimens
were counter stained with Hoechst 33342 and
finally mounted with mowiol.
Transmission electron microscopy
analysis
Specimen are washed in PHEM and
permeabilized with 0.05% thermanox triton.
aldehydblock ( 0.05M glycin in PHEM) and
proteinblock P ( 5%BSA + 0.1% CWFG in
PHEM) were performed before primary
antibody incubation for 3h. The secondary
antibody was incubated over night. Specimens
were post-fixed with 2%GA in A.d.. The signal
of the nanogold is than enhanced with silver
enhancement (Pharma). For generation of
contrasts the specimen are treated with 0.5%
OsO4 in a.d. and with Tannin 1% in 0.1M Cac.
The probes were finally dehydrated with
Isopropanol (70%>90%>100%). The cells
were than incubated with epon and finally
polymerized at 60°C.
Antibodies and Reagents
For Western Blotting analysis we used rabbit
anti TSC2 (1:2000), anti P-AKT (1:2000), anti
P-S6 (1:2000), anti LC3 (1:2000), anti P-ULK1
(1:2000), anti total AKT (1:2000), anti P-4EBP1 (1:2000) and mouse anti total S6
(1:2000). As secondary antibody sheep anti
rabbit (1:10000) and sheep anti mouse
(1:10000) were applied.
HeLa cells for immunofluorescence were
incubated with #291 anti-human CD107a
(LAMP-1) mouse monoclonal antibody (1:500),
Pharmingen 34201A. Goat anti-mouse Alexa
568 (1:500) was used as secondary antibody.
BisBenzimide H33342 trihydrochloride
(Hoechst 33342), Sigma, catalogue number
14533, was used for DAPI staining.
HeLa cells for transmission electron
microscopy were stained with mouse anti
LAMP1 (H4A3) diluted 1:40. nanogold goat
Lohmüller M
anti mouse Fab’ (Nanoprobes (#2002) Lot
#CG08R70) was used as secondary antibody.
The staining was conducted in 0.1M PHEM, all
antibodies and silver enhancement was
performed in WB (0.1% BSA-c in PHEM).
Cell diameter measurement
MEF cells were trypsinized and diluted in
DMEM to same dilution. Cells were mixed with
same amount of trypan blue gently and added
to the chamber ports of the Countess TM cell
counting chamber slide. The cell diameter was
measured with the Countess TM cell counter
instrument.
References
Hsu PP. The mTOR-regulated
phosphoproteome reveals a mechanism of
mTORC1-mediated inhibition of growth factor
signaling.Science. 2011; 1317-22.
Gomez MR. Varieties of expression of
tuberous sclerosis. Neurofibromatosis. 1988; 1
(5-6);330-338.
Manning BD. Spatial Control of the TSC
Complex Integrates Insulin and Nutrient
Regulation of mTORC1 at the Lysosome. Cell.
2014; 156, 771–785
Sehgal SN. Rapamycin (AY-22,989), a new
antifungal antibiotic. I. Taxonomy of the
producing streptomycete and isolation of the
active principle.J-Stage. 1975. 28(10):721-6
Teis D. p14MP1 MEK1 signaling regulates
endosomal traf c and cellular proliferation
during tissue homeostasis. J. Cell Biol.
175:861-868. http://dx.doi.org/ 10.1083/jcb.
200607025
12
Editor
Innrain 50-52
Centrum Chemie Biomedizin
6020 Innsbruck
Dear Editors,
Michael Lohmüller
7 March 2016
Medical University Innsbruck
Christoph Probst Platz
6020 Innsbruck
I’m honored to submit to you my Paper “The infinite mTORC1 signaling pathways
and potential treatment options for TSC patients.“
Our work described in this paper contributes to the actual field of mTORC1 and
TSC2 research. We addressed to shed new light on the pathways of mTOR signaling
to open new ways for potential treatments for tuberous sclerosis patients. The results
we present to you, that we can differentiate between various activation pathways of
mTOR, between straightforward and complex regulated actions. These can be further
used to analyze these proposed pathways to generate a better understanding of the
signaling processes.
Furthermore we found in transmission electron microscopy, that there is a tremendous
alteration of cell organelle amount in the TSC2KO cell line. The yield of
mitochondria is dramatically increased. This could also be further investigated to
analyze these unknown link of mTORC1 towards mitochondria. This is really
important in the development of hamartomas, from which the TSC patients suffer.
We hope to convince you, that our work will interest readers of your journal and that
the scientific world can use this informations to gain more knowledge about the field
of mTOR and tuberous sclerosis.
Sincerely,
Michael Lohmüller (B.Sc.)
Dear Authors,
Thank you for submitting your manuscripts to the Journal of Molecular Medicine
Innsbruck (JMMI). The manuscripts have now been seen by external reviewers that
are experts in this area.
Congratulations, the assement of the reviewers is in favour of publication in JMMI.
Your work is in principal accepted, provided several small changes (indicated with *)
can be addressed within the next two weeks.
We are looking forward seeing your improved manuscripts.
Please do not hesitate to contact me in case of questions
Best regards
David Teis
Specific comments for the authors:
Seyman: very good cover letter, but a sever mistake in the abstract. please fix that.
Ulli: overall very well written (for coverlet please see seyman’s coverlet). Please fix
the sizebars in your figures. they cannot be correct.
Hannah: also in general well written. however the labeling of the figures could better
(very difficult to read) and there is also a severe mistake at the end of the discussion.
please fix that.
general remarks:
Abstract:
A typical abstract is about 150 - 200 words (depends on the Journal:
First, you introduce the general biological problem.
Then you identify what is not known: what are the open questions
Next you describe what you have done.
Finally you conclude how your work has addressed these questions and move the
field forward.
cover letter:
is a somewhat extended version of the abstract.
Most importantly the cover letter should make the editor curious about your work and
highlight the noveltiy and unique character of your work and how it moves the field
beyond the current state of the art.
The cover letter represents your gateway towards the review process.
Most papers are rejected without review at this stage.
Hence a good and strong cover letter can make a big difference.
I’m attaching a recent example from my lab.