Download STSM Scientific Report Short Term Scientific Missions COST Action

STSM Scientific Report
Short Term Scientific Missions
COST Action BM 1007
Adi Efergan
Department of Cell and Developmental Biology
Sackler Faculty of Medicine
Tel-Aviv University
Ramat-Aviv 61390 TEL AVIV, P.O.B 39040 ISRAEL
Phone +972 (52) 6164488
Email [email protected]
Purpose of the STSM:
Cutaneous mastocytosis (CM) is characterized by abnormal proliferation and
infiltration of mast cells (MCs) within the skin and is associated with both local and
systemic symptoms. Unlike the systemic form of mastocytosis that is mostly linked
with activating mutations (i.e. (D816V)) of the stem cell factor (SCF) receptor c-kit
that is important in MC survival, CM patients often lack codon 816 mutations in c-kit.
In these cases the genetic background of the kit negative CM patients is unknown.
A number of case reports concerning c-kit negative patients describe a form of
CM that is linked with morphological alterations manifested as MCs that contain giant
cytoplasmic granules. Intriguingly, a similar phenotype was recently observed in our
laboratory as a result of hyper activation of the small GTPase Rab5. These findings
thus raise the possibility that the gene (s) that are mutated in CM patients whose MCs
contain giant granules are involved in the control of the secretory granules (SGs) size
and most likely reside along the Rab5 regulated pathway.
The intention of this study is therefore to test the feasibility of our hypothesis
and investigate if there is a link between Rab5 and the phenotype observed in this
subgroup of CM patients.
We anticipate that the results emerging from this study will lay the ground for
improving the treatment of these patients and will be an instrumental for the future
understanding of MCs activation in the course of pathological conditions.
Since our original observation on the formation of giant SGs in MCs by active
Rab5 was made in RBL-2H3 cells, a rat MC line, we are first interested in confirming
our results in non transformed human MCs, hence demonstrate that expression of
active Rab5 indeed affects the size of SGs. Therefore, my goal was to test a number of
models of human MCs. In particular I was interested to learn the techniques of
culturing human MCs from peripheral stem cells, to determine the best conditions for
genetic manipulations of human MCs and finally to express active Rab5 mutants and
test the impact on cell growth and survival as well as on c-kit internalization, in view
of the fact that .Rab5 is a key regulator of endocytic pathways that is likely to affect ckit trafficking, Therefore, we were interested to analyze the fate of c-kit in MCs
displaying giant SGs.
Description of the work carried out during the STSM:
I stayed at the Department of Dermatology and Allergy of CharitéUniversitätsmedizin, Berlin, from 28 of April 2014 until the 1 of June 2014. During
this period of time I trained how to set up a human MC culture from CD34+ stem
cells. In parallel, I also attempted to establish conditions for genetic manipulations of
cultured human skin derived MCs and LAD2 cells. Setting up conditions for
transfection of LAD2 cells was successful, whereas manipulation of cultured skin
MCs will require further characterization.
Using the LAD2 cells, I was able to demonstrate that NPY-mRFP, which we
used as reporter for exocytosis in RBL cells, is also a valuable reporter for exocytosis
and SGs in studies with LAD2 cells. The reporter can be expressed in LAD2 cells and
it is targeted to the SGs (Fig. 1). I could also show that Rab5 that was introduced into
the cell fused to GFP colocalized with NPY-mRFP on the SGs (Fig. 1). Finally, and
most importantly, my analyses revealed that expression of a constitutively active (CA)
GTP-locked Rab5 mutant induces giant SGs in LAD2 cells, consistent with our results
in RBL cells (Fig. 1).
Fig.1: Rab5 regulates the SGs size. LAD2 cells
were transiently co transfected with NPY-mRFP
and with either GFP, GFP-WT Rab5 or GFP-CA
Rab5 cDNAs. The cells were seeded and grown
on glass coverslips for 24 hr, fixed and analysed
by confocal microscopy. The arrowhead points
to the large structures formed (giant SGs).
I have also compared the survival of LAD2 cells transfected with different
mutants of Rab5 by Annexin V/7AAD staining, in order to investigate whether
expression of a mutated Rab5 might provide a survival advantage. However, during
the tested time period (4 days), no significant differences in cell survival were noted. I
envision that to address this question, we will have to stably transfect the cells to be
able to monitor their survival for longer time periods.
I next analyzed the impact of Rab5 activation on cell surface expression of c-kit,
by FACS analysis using LAD2 cells transfected with different Rab5 mutants as a
function of their time of exposure to SCF.
The summary of two independent experiments is presented (Fig. 2) revealing a
complex regulation of c-kit trafficking by Rab5 that would need further clarification.
My results show that in cells expressing CA Rab5, a larger fraction of the
receptor remains at the cell surface. We therefore expect that CA Rab5 expression
will have an impact on c-kit signaling. We postulate that Rab5 presumably regulates
not only the internalization of c-kit from the plasma membrane to early endosomes,
but also its subsequent transport to the late endosomes and lysosomes for degradation.
Therefore, there is a genuine need to monitor the route of internalized c-kit with
reference to endocytic markers and to evaluate the impact on its elicited signaling.
Fig. 2: Effect of Rab5 on SCF-induced c-Kit internalization. LAD2 cells were transiently
transfected with either GFP, GFP-WT Rab5, GFP-CA Rab5 or GFP-CN Rab5 cDNAs. The cells
starved for 16 h in DMEM, then stimulated with 100 ng/ml SCF for the indicated times, and
surface c-Kit expression levels were analyzed by flow cytometry.
Future collaboration with host institution:
In collaboration with the laboratory of Prof. Marcus Maurer and Dr. Frank
Siebenhaar at the Department of Dermatology and Allergy in Berlin, we established a
methodology that allows us to screen and identify the subset of CM patients that
display giant SGs in their skin MCs. We stained sections of biopsies derived from both
c-kit positive and c-kit negative CM patients by toluidine blue to evaluate the MCs
burden. Next, MCs present in skin biopsies of CM patients were stained with anti
tryptase and analyzed by confocal microscopy at a resolution that allows
morphometric analyses of the SGs size.
We hypothesize that a genetic connection exists between this subtype of CM and
the formation of giant SG, whereby the gene(s) mutations that cause the formation of
giant SGs in the MCs are also the underlying cause for CM in these patients. Our aim
is to identify the underlying mutations in the MCs of these patients that display such
phenotypic alteration.
In order to characterize the mutation that causes enlarged SGs in CM cells, we
will analyze DNA samples that were taken from the relevant patients for expression of
mutations in Rab5 or Rab5 regulating genes. An unbiased screen will follow this step
by exome sequencing of the DNA samples.
Identification of the underlying gene mutation(s) in this sub group of CM patients
will lay the ground for novel therapeutic approaches for this class of patients.