89,075 research outputs found
Studies on Memo, an important ErbB2 receptor-mediated component of the cellular migratory machinery
No full textThe ErbB2 receptor tyrosine kinase has been shown to play an important role in cancer cell
motility and metastases formation. This receptor is often overexpressed in human tumors of
diverse origins, including breast and ovarian cancer. Individuals with ErbB2 over expressing
tumors have shown poor clinical outcome.
Our studies are focused on signaling molecules that interact with autophosphorylated tyrosine
residues of the cytoplasmic tail of the receptor. Two of the sites, Tyr 1201 (YC) and Tyr 1227
(YD) are fully able to restore the migratory phenotype of breast carcinoma cells. Studies of
the functional role of ErbB2 phosphorylation sites identified PLCγ1 as an interacting partner
of the YC autophosphorylation site, and Memo (Mediator of ErbB2-driven cell Motility) as a
binding partner of the YD site that is required for ErbB2 induced cell motility.
Memo is encoded by a unique gene that is found in all branches of life, from bacteria to
humans. Memo has no characterized domains, nor does it have obvious catalytic activity.
Various approaches were used to position Memo in a signaling pathway and to uncover its
biochemical function. Memo was initially detected based on its important role in ErbB2-
induced cell motility. In fact, tumor cells with a specific knock-down (KD) of Memo failed to
grow microtubules in response to Heregulin (HRG)-induced ErbB2 activation and were
impaired in their migration.
Cell migration proceeds in distinct steps. In response to a chemotactic stimulus, cells extend
protrusions at the front that help in attachment. This is followed by contraction of the cell
body and tail detachment at the rear allowing movement in the direction of the ligand. The
initial event in the process is sensing of the ligand in response to activation of cellular
receptors like EGFR or ErbB2. Their activation initiates signaling pathways that lead to
polymerization of new actin at the leading edge, which is necessary for generating the
protrusive force allowing migration. An important goal of my thesis work has been to
investigate the step(s) of the migratory process that require Memo.
In the first study, we explored migration using Dunn chambers and analyzed the chemotactic
response of tumor cells in a shallow gradient of ligand. By tracing HRG-stimulated cell
migration in time-lapse video microscopy, we found that Memo or PLCγ1 KD strongly
impairs cell directionality, reflecting an important role for Memo and PLCγ1 in orchestrating
directional cell migration. We also demonstrated that depletion of Memo or PLCγ1 resulted in
very similar phenotypes, with a strong impairment of HRG-induced cytoskeletal organization.
To gain more insight into Memo’s function, we carried out a Yeast-2-Hybrid (YTH) analysis
and found a number of interesting new partners of interaction for Memo. Of particular interest
is the small protein cofilin, one of the major cellular actin severing and depolymerizing
factors that is known to have an essential role in directional sensing during chemotaxis. This
interaction was confirmed in vitro using recombinant proteins and in vivo in coimmunoprecipitation
experiments where Memo was detected in complexes with cofilin,
ErbB2 and PLCγ1. Interestingly, we also found that HRG-induced PLCγ1 phosphorylation
was decreased in Memo KD cells, suggesting that Memo regulates PLCγ1 activation.
Furthermore, by introducing GFP-tagged cofilin into control, Memo or PLCγ1 siRNA
transfected breast tumor cells, we showed that HRG-induced recruitment of GFP-cofilin to
lamellipodia is impaired in Memo- and in PLCγ1 KD cells, suggesting that both proteins lie
upstream of cofilin in models of ErbB2-driven tumor cell migration. Finally, we examined the
effect of Memo on cofilin binding and severing/depolymerizing properties. In vitro F-actin
binding assays showed that Memo does not impair cofilin binding to F-actin, and revealed
that Memo is a novel F-actin binding protein. In vitro F-actin depolymerization assays
indicated that Memo promotes cofilin depolymerizing/severing activity. Altogether, these
data suggest a novel role for Memo during the migratory process and its implication in the
regulation of actin dynamics through cofilin binding.
In the second study, we used two different Memo-defective cellular models to examine
Memo’s function in more detail. We demonstrated that inhibition of Memo impairs activation
of a number of signaling molecules including Src, Shc, ERK and PLCγ1. We also provide
evidence that Memo interacts with the three Shc isoforms, p46shc, p52shc, and p66shc, and
showed that Shc is required for Memo binding to the ErbB2 receptor. Control and Memodeficient
cells were also scored for their migration and adhesion properties. These assays
indicated that Memo is important in both cell migration and adhesion processes. Also,
morphological and biochemical analyses of control and Memo-deficient cells suggested that
Memo is involved in focal adhesion organization and rear cell deadhesion during the
migratory process.
Altogether, these two studies revealed important roles for Memo at different steps of cell
migration and metastasis, making it a potential interesting target for cancer therapy.
Genetic approaches in model organisms have been important for gaining insight into the
function of evolutionarily conserved proteins. To position Memo within a genetic network,
experiments in the model organism S. cerevisae that lends itself to rapid genetic screening
were performed. We investigated cellular localization of Memo in yeast and found that Memo
is located in the nucleus and cytoplasm of the cell. A S. cerevisae memo Δ strain has been
generated and is viable. Considering the role of Memo in the microtubule and actin networks
that we described in mammalian cells, we examined the memo Δ strain for defects in different
cytoskeletal dynamics. No significant effect was observed. We also performed a Synthetic
Lethal Screen of genetic interactions between a memo Δ strain and an ordered array of 4700
Yeast strains containing non-essential gene deletions. This analysis revealed a limited number
of synthetic interactions. Lethality was observed in combination with the plc1Δ strain. PLC1
encodes for the unique isoform of phosphatidylinositol-specific phospholipase C of S.
cerevisiae. The results are intriguing and exciting considering the data obtained in the
mammalian models; in fact, we demonstrated that Memo and PLCγ1 interact with ErbB2
autophosphorylation sites and are essential for directional migration. We also showed that
Memo is found in a complex with PLCγ1 and ErbB2 and that Memo is likely contributing to
PLCγ1 activation. We hypothesize that in Yeast, Memo and PLC1 act in the same or in
distinct but related pathways, and suggest that the connection between PLC and Memo
induced-pathways is also conserved through evolution
[Memo from M. F. Hass, Lieutenant Colonel, regarding over-registration under Executive Order No. 54]
No full textA memo sent from Lieutenant Colonel M. F. Hass regarding over-registration due to Exclusion Order No. 54. The memo details the number of evacuees per day between May 12 and May 14, 1942, including the assembly center destination.The War Relocation Authority (WRA), together with the Wartime Civil Control Administration (WCCA), the Civil Affairs Division (CAD) and the Office of the Commanding General (OFG) of the Western Defense Command (WDC) operated together to segregate and house some 110,000 men women and children from 1942 to 1945. The collection contains documents and photographs relating to the establishment and administrative workings of the (WDC), the (WRA) and the (WCCA) for the year 1942
Analysis of in vivo functions of Memo in embryonic and mammary gland development
No full textStudies from our lab recently led to the discovery of Memo (mediator of ErbB2-driven cell motility), a novel 297 amino acid protein shown to be required for ErbB2- and other receptor tyrosine kinase-driven cell motility in breast tumor cells. Inhibition of Memo expression had consequences on the microtubule network which could not grow towards the periphery of the cells upon heregulin (a ligand activating ErbB2/ErbB3 and ErbB2/ErB4 heterodimers) stimulation. It also had consequences on the actin cytoskeleton, since more actin stress fibers were seen.
To explore the biological function of Memo, and in order to check if Memo also plays a role in in vivo cell migration events, we generated animal models deficient for Memo. We found that Memo is expressed ubiquitously in adult organs as well as in organs of the developing embryo. Unexpectedly, we did not see any defect in migration in vivo, despite the presence of a lot of migrating events during development like gastrulation or migration of the neural crest derivatives or of the somitomeres. Instead, we found that Memo seems to play a role in vascular integrity, as demonstrated by the presence of hemorrhages and the dilated small vessels in the Memo deficient embryos. This leads to the death of Memo deficient embryos after 13 days of embryonic development.
To study the in vivo role of Memo in the lactating mammary gland, we generated mice deficient for Memo in luminal alveolar epithelial cells (the cells that produce and secrete milk during lactation). We measured a decrease in the weight of pups from Memo deficient mothers, indicating that they were unable to correctly nurse them. The weight of the mammary gland itself was smaller in the Memo deficient females compared to control females. By histological analyis we saw the abnormal presence of shed cells in the lumen of Memo deficient glands in the first days of lactation. We saw a progressive loss of alveoli (formed by epithelium) which were replaced by adipocytes. Increased apoptosis (controlled
cell death) was measured in the Memo deficient glands. Consistent with this apoptosis seen at the histological level, we could see an increase in the levels of pro-apoptotic P-Stat3 and Bax at protein level. We also could see improper localization of the adherens junction proteins E-cadherin and ß-catenin in the Memo deficient mammary glands. We therefore propose that in the mammary gland Memo plays a role in epithelial cell-cell adhesion, and that if this role is not properly achieved, the cells undergo apoptosis and are shed in the lumen of alveoli which progressively disappear. This leads to improper feeding of the pups
Recommended from our members
[Memo from Alfred F. Hurley]
No full textA memo from Alfred F. Hurley inviting the receiver to an executive lunch briefing on the North Texas Regional institute for Educators on the Visual Arts, now known as the North Texas Institute for Educators on the Visual Arts. Attached document is parking instructions
[Memo from Lieutenant Colonel M. F. Hass, Civil Affairs Division, regarding the evaucation schedule for Exclusion Order No. 92]
No full textA one page memo from Lieutenant Colonel M. F. Hass, Civil Affairs Division, with an adjusted evacuation schedule related to Exclusion Order No. 93. Due to over-registration in the Sacramento area, the evacuation schedule was revised. The memo includes the number of people to be evacuated between May 27 and May 29, 1942.The War Relocation Authority (WRA), together with the Wartime Civil Control Administration (WCCA), the Civil Affairs Division (CAD) and the Office of the Commanding General (OFG) of the Western Defense Command (WDC) operated together to segregate and house some 110,000 men women and children from 1942 to 1945. The collection contains documents and photographs relating to the establishment and administrative workings of the (WDC), the (WRA) and the (WCCA) for the year 1942
[Memo from Lieutenant Colonel M. F. Hass, Civil Affairs Division, about the evacuation schedule from Sacramento County]
No full textA one page memo from Lieutenant Colonel M. F. Hass, Civil Affairs Division, with an adjusted evacuation schedule related to Exclusion Order No. 93. The memo states that the adjusted schedule was due to requests from Japanese families to be reunited in the Sacramento Assembly Center. The evacuation dates are from May 27 to May 29, 1942.The War Relocation Authority (WRA), together with the Wartime Civil Control Administration (WCCA), the Civil Affairs Division (CAD) and the Office of the Commanding General (OFG) of the Western Defense Command (WDC) operated together to segregate and house some 110,000 men women and children from 1942 to 1945. The collection contains documents and photographs relating to the establishment and administrative workings of the (WDC), the (WRA) and the (WCCA) for the year 1942
Faculty memo
No full textMemo to TCU faculty from Thomas F. Richardson, Dean of Students, about student veterans
[Memo from Lietnenant Colonel M. F. Hass, Civil Affairs Division, regarding the destination for Exclusion Order No. 98]
No full textA one page memo from Lieutenant Colonel M. F. Hass, Civil Affairs Division, that declares the destination for evacuees affected by Exclusion Order No. 98. The evacuees from all three movements were sent to the Portaln Assembly Center in Oregon.The War Relocation Authority (WRA), together with the Wartime Civil Control Administration (WCCA), the Civil Affairs Division (CAD) and the Office of the Commanding General (OFG) of the Western Defense Command (WDC) operated together to segregate and house some 110,000 men women and children from 1942 to 1945. The collection contains documents and photographs relating to the establishment and administrative workings of the (WDC), the (WRA) and the (WCCA) for the year 1942
The ErbB2 receptor and breast carcinoma cell migration : memo is a novel mediator of cell motility
No full textThe ErbB family of receptor tyrosine kinases play important role in normal physiological processes occurring during development; moreover, their deregulated expression has been implicated in human cancer. Cancer patients, whose tumors have alterations in ErbB1 or ErbB2, tend to have a more aggressive disease associated with parameters predicting a poor clinical outcome, including tumor metastases. For tumors to metastasize, the cells have to possess specific characteristics, including the ability to migrate and to invade the surrounding basal membrane. The role of the Neu/ErbB2 receptor in cancer cell migration is the major topic of this thesis. The Neu/ErbB2 receptor is often overexpressed in different human tumors, including breast and ovarian tumors. Clinical and in vitro studies revealed that Neu/ErbB2 plays important functions in tumor cell motility. Upon ErbB receptors activation via ligand-induced dimerization, receptors autophosphorylate specific tyrosines in the carboxy domain leading to activation of downstream signaling cascades, including the mitogen-activated protein kinase (MAPK) and the phosphatidylinositol-3-kinase (PI3K) pathways. These pathways, which are known to be important for cell migration, are involved in actin cytoskeleton remodeling, leading to formation of lamellipodia and actin stress fibers. In this work we used T47D breast carcinoma cells expressing Neu/ErbB2 add-back mutants harboring none or only one of the five major autophosphorylation sites, to study the contribution of individual Neu/ErbB2 tyrosine autophosphorylation sites in cell migration. We showed that activation of MAPK and PI3K in T47D cell failed to induce efficient cell motility in the absence of the Neu/ErbB2 tyrosines 1201 or 1227 phosphorylation. Moreover, we present evidence that efficient, long-term cell migration depends upon ongoing transcription and translation. Signaling downstream of tyrosine 1201 and 1227 is required for de novo synthesis of RNA and protein involved in cell migration. Further investigation of the function of these two tyrosines led to the identification of a novel protein that specifically interacts with the phosphorylated tyrosine 1227. We called this new protein Memo for mediator of ErbB2-driven cell motility. Memo does not bind directly to the phosphorylated tyrosine 1227 of the Neu/ErbB2 receptor, but very likely via the adaptor molecule Shc. Memo is required for ErbB2-driven breast carcinoma cell migration, because its downregulation leads to decreased motility of cells expressing the receptor with the tyrosine 1227. Interestingly, we found that Memo is not only required for migration downstream of the ErbB receptors, but it may be a general mediator of growth factor-induced breast carcinoma cell migration. Cell migration is a multistep process and we further defined at which step Memo is required. We found that upon Neu/ErbB2 activation, wild type cells, but interestingly also Memo-deficient cells form actin stress fibers and extend lamellipodia. However, Memo-deficient cells are not able to extend microtubules toward the cell cortex. There is increasing evidence that not only the actin cytoskeleton but also the microtubule cytoskeleton plays a crucial role for cell migration. For instance, microtubules are required for the polarization of the cells and also for the transport of proteins required for motility to the cell leading edge. Further studies have to be done to understand the exact role of Memo in microtubule outgrowth and its contribution to cell motility.
In summary, the work presented in the thesis shows the identification of a novel protein, Memo, which is required for breast carcinoma cell migration. We propose that Memo controls cell motility by transmitting extracellular chemotactic signals to the microtuble cytoskeleton
A new idea of coffee: the concept of cocktail cappuccino
No full textIt’s not easy to identify and define the role of design in the extremely liquid society in which we are
living. It should assume the politic role of mediator between different fields and cultures, stressing ever
more the importance of using the lateral thinking to solve problems and find solutions instead of a
horizontal one. Moreover, design should help people to open their mind, looking for links and
connections invisible at first sight.
In this phase of the 21st century characterized by several changes in people’s lifestyles, jobs and
working modalities, we are witnessing an incredible growth in the desired quality and consideration
people give to what they eat and drink. In fact, Food Design is now recognised as one of the main
design sectors almost equal to Industrial and Interior branches. Whole grain products for food and
gourmet coffees for beverages are becoming new standards in the culinary world.
But what is design doing in this field? On the one hand, it is already used in several applications like
Design With Food, Design For Food or Eating Design if we consider the design of food itself, the
design of products useful to cook or the design of unusual eating situations. On the other one it is
backing the creation and expansion of new forms of drinks by using young people as first promoters
and consumers. More specifically, design is paying particular attention to the changing trend from hot
to cold coffee all over the world: Food, Industrial and interior Design merge together for the recreation
of the whole coffee beverage experience. The analysis of processes, modalities and environments
where foodstuffs and beverages -with a deep focus on cappuccino- are elaborated, distributed and
consumed has led to the creation of new formats of alimentary products characterized by new
aesthetic, communicative and representative values.
The following paper wants to show the evolution and spread in cold coffee consumption around the
world by investigating which are the new social and inclusive spaces that can be considered as new
focal points to drink specialty coffees. From the United States to Asia Pacific and Europe: the future
coffee will not be the same that we are used to drink today.
Nowadays coffee is not only a prime morning necessity, a whim or a habit, but it’s a real pretext for
have a dialogue and a possible reason for discussing. In recent years, several new key drivers are
changing people’s perception about one of the most popular and consumed drinks in the world:
geographical context, seasonality and quality emerged as new pillars that users consider in coffee
selection. At the same time, the places in which this beverage is consumed are changing too: the
borders among bars, hotels and restaurants are disappearing.
This study is based on a deep analysis conducted at the Design School of the Politecnico di Milano
(Italy) which involved also two leader companies in coffee machines and technical components
production. The convergence of interdisciplinary and transversal skills coming from management,
engineering and design fields helped in the modelling of a research methodology which specifically
investigated the cold cappuccino sector from a completely different perspective. This kind of approach
encourages and supports strategic innovations, through which it’s possible to imagine the cappuccino
more like a spectacular cocktail than a simply drink
- …
