What molecular processes are involved in pseudopodial extension?

What molecular processes are involved in pseudopodial extension?

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I am curious as to the processes and mechanisms involved in the extension of pseudopodia in amoeba. How does the cell know and control the direction and extent of pseudopodia formation at a molecular level? I am not particularly interested in the taxes that amoeba recognize, but rather the mechanism of response to these taxes.

The extracellular cue signal must be relayed to the cell by Rho family of GTPases, like in the case of filopodia and lammelipodia. This causes local actin polymerization leading to extension of pseudopodium.

For a casual reference you can check the wikipedia page on Rho family of GTPases. Cell biology books like MBOTC also have information on mechanism of cytoskeletal dynamics.

What molecular processes are involved in pseudopodial extension? - Biology

Nonmotile cells extend and retract pseudopodia-like structures in a random manner, whereas motile cells establish a single dominant pseudopodium in the direction of movement. This is a critical step necessary for cell migration and occurs prior to cell body translocation, yet little is known about how this process is regulated. Here we show that myosin II light chain (MLC) phosphorylation at its regulatory serine 19 is elevated in growing and retracting pseudopodia. MLC phosphorylation in the extending pseudopodium was associated with strong and persistent amplification of extracellular-regulated signal kinase (ERK) and MLC kinase activity, which specifically localized to the leading pseudopodium. Interestingly, inhibition of ERK or MLC kinase activity prevented MLC phosphorylation and pseudopodia extension but not retraction. In contrast, inhibition of RhoA activity specifically decreased pseudopodia retraction but not extension. Importantly, inhibition of RhoA activity specifically blocked MLC phosphorylation associated with retracting pseudopodia. Inhibition of either ERK or RhoA signals prevents chemotaxis, indicating that both pathways contribute to the establishment of cell polarity and migration. Together, these findings demonstrate that ERK and RhoA are distinct pathways that control pseudopodia extension and retraction, respectively, through differential modulation of MLC phosphorylation and contractile processes.

Molecular mechanisms of life- and health-span extension: role of calorie restriction and exercise intervention

The aging process results in a gradual and progressive structural deterioration of biomolecular and cellular compartments and is associated with many pathological conditions, including cardiovascular disease, stroke, Alzheimer's disease, osteoporosis, sarcopenia, and liver dysfunction. Concomitantly, each of these conditions is associated with progressive functional decline, loss of independence, and ultimately disability. Because disabled individuals require care in outpatient or home care settings, and in light of the social, emotional, and fiscal burden associated with caring for an ever-increasing elderly population, research in geriatric medicine has recently focused on the biological mechanisms that are involved in the progression towards functional decline and disability to better design treatment and intervention strategies. Although not completely understood, the mechanisms underlying the aging process may partly involve inflammatory processes, oxidative damage, mitochondrial dysfunction, and apoptotic tissue degeneration. These hypotheses are based on epidemiological evidence and data from animal models of aging, as well as interventional studies. Findings from these studies have identified possible strategies to decrease the incidence of age-related diseases and delay the aging process. For example, lifelong exercise is known to extend mean life-span, whereas calorie restriction (CR) increases both mean and maximum life-span in a variety of species. Optimal application of these intervention strategies in the elderly may positively affect health-related outcomes and possibly longevity. Therefore, the scope of this article is to (i) provide an interpretation of various theories of aging from a "health-span" perspective (ii) describe interventional testing in animals (CR and exercise) and (iii) provide a translational interpretation of these data.


HGF (hepatocyte growth factor) is secreted by cells of mesenchymal origin and acts on epithelial cells, stimulating cell division, disrupting cell—cell adhesion and inducing cell motility or scattering, as well as tubule morphogenesis. HGF activates its receptor, c-Met, encoded by the c-met proto-oncogene, leading to receptor autophosphorylation, interaction with SH2 (Src homology 2)-domain-containing proteins, such as Grb2 (growth-factor-receptor-bound protein 2), Gab1 (Grb2-associated binding protein 1), Src and Stat3 (signal transducer and activator of transcription 3), and activation of complex downstream signalling pathways (Birchmeier et al., 2003 ). Deregulated expression of c-Met signalling via expression of the c-Met cytoplasmic domain under control of a tpr (translocated promoter region) generates the Met oncogene and leads to cellular transformation and tumorigenicity (Park et al., 1986 Bottaro et al., 1991 ). Overexpression, increased tyrosine phosphorylation and activating mutations of c-Met are associated with tumour progression (Comoglio and Boccaccio, 2001 Ma et al., 2003 ). HGF/c-Met autocrine loops are present in various tumours, and co-expression of HGF and its receptor results in the acquisition of metastatic potential (Rong et al., 1994 Ferracini et al., 1995 Tuck et al., 1996 Wong et al., 2001 ). In HGF transgenic mice, constitutive expression of c-Met via either paracrine or autocrine signalling leads to tumorigenesis and promotes metastasis (Horiguchi et al., 2002 Yu and Merlino, 2002 Gallego et al., 2003 ). Decoy c-Met receptors and extracellular c-Met domains (Sema) that interfere with HGF binding and c-Met dimerization have shown potential as anti-cancer agents (Kong-Beltran et al., 2004 Michieli et al., 2004 ).

Targeting of the tpr—Met chimaera to the plasma membrane by a c-Src myristoylation signal enhances cellular transformation and induces cellular protrusions, implicating localized c-Met activation in motility induction (Kamikura et al., 2000 ). Pseudopodia of motile cells represent polarized cell surface domains whose protrusion is the initial step in cellular displacement, and whose subsequent stabilization via substrate adhesion determines the directionality of cell movement (Lauffenberger and Horwitz, 1996 Mitchison and Cramer, 1996 Nabi, 1999 ). Autocrine activation of the EGFR (epidermal growth factor receptor) has been shown to lead to increased persistence of motility in a mammary carcinoma cell line (Maheshwari et al., 2001 ). However, a hallmark of tumour cell motility is its random nature. The demonstration that autocrine c-Met activation is responsible for pseudopodia formation in the MSV-MDCK-INV (invasive Moloney-sarcoma-virus-transformed Madin—Darby canine kidney) variant provided the first indication of a role for intrinsic autocrine signalling mechanisms in pseudopod formation (Vadnais et al., 2002 ).

HGF-induced scattering in MDCK cells is Rac- and Cdc42-dependent and inhibited by expression of DA (dominant active)-RhoA. DN (dominant negative)-RhoA and inhibition of ROCK (Rho kinase) do not prevent HGF stimulation of lamellipodia formation in MDCK cells and regulate formation of focal contacts and stress fibres (Ridley et al., 1995 Kodama et al., 2000 Royal et al., 2000 ). However, expression of oncogenic Ras in MDCK cells leads to reduced Rac and enhanced Rho activity that is associated with epithelial—mesenchymal transition (Zondag et al., 2000 ). MSV-MDCK-INV cells were selected from MSV-transformed MDCK cells for their enhanced invasive capacity, and their multiple β-actin-rich pseudopodial domains are blebbed and morphologically distinct from lamellipodia and filipodia (Le et al., 1998 Nguyen et al., 2000 ). MSV-MDCK-INV pseudopodia are induced by RhoA activation, and inhibition of the downstream ROCK regulates pseudopodial mRNA translocation and transforms these protrusive domains into extended lamellipodia (Jia et al., 2005 ). An alternative Rho/ROCK-dependent, non-proteolytic, blebbed mode of cellular invasion has been described (Sahai and Marshall, 2003 Wolf et al., 2003 Wilkinson et al., 2005 ). In the present study we show that cellular blebbing and pseudopodial protrusion of MSV-MDCK-INV cells are both Rho/ROCK-dependent, require autocrine c-Met activation and are mediated by a p38 MAPK (p38 mitogen-activated protein kinase) signalling pathway. Autocrine c-Met regulation of local Rho/ROCK/p38 MAPK-directed cellular blebbing and pseudopodial protrusion suggests that these two processes implicated in tumour cell motility are related.


Reagents and cells

Panobinostat (Novartis Pharmaceuticals), romidepsin (Celgene), ABT-737 (Abbott Laboratories), AMP-4 (Amgen), N-hydroxysuccinimidobiotin (NHS-biotin Sigma), and thiazole orange (Sigma) were provided or purchased as indicated. Murine TPO was synthesized at the Walter and Eliza Hall Institute (Parkville, Australia). Anti–P21-activated kinase 1 (anti-PAK1), anti–phospho-myosin light chain 2 (anti-pMLC2), and anti-MLC2 were purchased from Cell Signaling Technology anti-Rac1 and anti-CDC42 were from Becton Dickinson and anti-RhoA was from Santa Cruz Biotechnology. WR PAK18 was purchased from EMD Chemicals avidin and biotin solutions were purchased from Dako and a tyramide signal amplification biotin kit was purchased from Perkin Elmer. Meg-01 cells were purchased from DSMZ and cultured in 10% RPMI 1640 supplemented with 10% fetal calf serum, penicillin/streptomycin, and l -glutamine. Murine megakaryocytes were derived from fetal liver cells harvested from E13.5-14.5 pregnant C57BL/6 mice and plated out at 5 × 10 5 cells/mL in StemPro-34 medium (Invitrogen) supplemented with murine TPO (100 ng/mL) at 37°C with 5% CO2 for 3-5 days.

All animal studies were approved by the Peter McCallum Cancer Center Animal Experiment Ethics Committee. C57BL/6 mice 8-12 weeks of age were purchased from the Walter and Eliza Hall Institute and injected with either panobinostat 10 mg/kg or romidepsin 1 mg/kg intraperitoneally (IP) daily, either continuously or on an alternate weekly schedule. After confirmation of TCP with both drugs on the continuous daily IP schedule, AMP-4 (20 μg/kg) was administered IP in combination with both drugs, with one dose given on the same day that HDACI treatment was started (day 0) and another on day 6. For the Bak −/− Bax −/− experiments, irradiated wild-type C57BL/6 mice were reconstituted with Bak −/− Bax −/− fetal liver cells, as described previously. 28 Briefly, wild-type or Bak −/− Bax +/− C57BL/6-CD45.2 mice were intercrossed and day-13.5 pregnant mice were killed, embryos removed, and fetal livers dissected. Genomic DNA was extracted from embryonic tissue and subjected to polymerase chain reaction amplification to determine genotype. Fetal-liver cell suspensions were prepared in phosphate-buffered saline (PBS), and 2 × 10 6 cells were injected into lethally irradiated C57BL/6-CD45.1 mice. To prevent infections, the transplanted animals were provided with water containing neomycin (Sigma). After stable reconstitution of their hematopoietic system (8 weeks later), donor contribution to peripheral blood leukocytes was analyzed by flow cytometry. Only mice showing 90%-98% engraftment were used in the experiments. 28 C-mpl–knockout mice were a gift from Prof Warren Alexander (Walter and Eliza Hall Institute). For all experiments, at least 15 mice per cohort were used, with 3 mice eye-bled at each time point, and experiments were repeated at least 3 times.

Platelet clearance analysis and reticulated platelet staining

Mice were injected intravenously twice, 1 hour apart, with 600 μg of NHS-biotin in PBS and 10% dimethyl sulfoxide. At various time points, peripheral blood was isolated from the tail, and 1 μL was washed twice in PBS and 10mM EDTA (ethylenediaminetetraacetic acid). Platelets were stained with phycoerythrin-conjugated rat anti–CD41 (Becton Dickinson) and allophycocyanin-conjugated streptavidin (APC-A) (Becton Dickinson) for 30 minutes on ice. Samples were washed again and incubated with 0.125μg/mL of thiazole orange, which has increased uptake in high-RNA–containing platelets, staining the younger, reticulated platelet fraction, for 90 minutes. Flow cytometry was then performed on an LSR flow cytometer (Becton Dickinson). By plotting the number of biotinylated platelets against time, an estimate of the life span was obtained by linear extrapolation, while the number of thiazole orange–positive, nonbiotinylated platelets provided an estimate of new platelet production. All experiments were performed at least 3 times.

TPO analysis

After collection of blood from treated mice, TPO levels were measured by enzyme-linked immunosorbent assay using the RayBio TPO ELISA kit (RayBiotech) according to the manufacturer's instructions.

Proplatelet formation assay

Fetal liver cells were harvested from E13.5-14.5 pregnant C57BL/6 mice, and plated out at 5 × 10 5 cells/mL in StemPro-34 medium (Invitrogen) supplemented with murine TPO (100 ng/mL) at 37°C with 5% CO2 for 3 days. Cells were subjected to a 3% (3 mL) and a 1.5% (3 mL) bovine serum albumin (BSA) gradient at 1g for 40 minutes at room temperature. Megakaryocytes were harvested from the lowest 800 μL to 1 mL and plated out at 1000 cells/100 μL in 96-well plates for 3 days with 100 ng/mL of TPO in combination with panobinostat or romidepsin in duplicate. Proplatelet-displaying megakaryocytes were defined as cells exhibiting one or more filament-like protrusions at least the diameter of the cell, counted under an inverted microscope, and the percentage of megakaryocytes with proplatelets was calculated. All experiments were performed at least 3 times.

Western blot analysis

Platelets were isolated from blood of mice by eye bleeding into 0.1 volume of Aster Jandl anticoagulant (85mM sodium citrate dihydrate, 69mM citric acid anhydrase, and 10mM glucose). Mouse platelet–rich plasma was obtained by centrifugation at 125g for 8 minutes. Platelets were washed by 2 sequential centrifugations at 860g for 5 minutes in wash buffer (140mM NaCl, 5mM KCl, 12mM sodium citrate, 10mM glucose, and 12.5mM sucrose, pH 6.0), and resuspended in resuspension buffer (10mM HEPES [N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid], 140mM NaCl, 3mM KCl, 0.5mM MgCl2 hexahydrate, 0.5mM NaHCO3, and 10mM glucose, pH 7.4). C57BL/6 fetal liver cells were plated out with TPO (100 ng/mL) and treated with 10nM panobinostat or 10nM romidepsin on day 3. On day 5, the cells underwent BSA gradient separation, as described in “Proplatelet formation assay,” and megakaryocytes were isolated and washed 3 times in PBS. Meg-01 cells were treated with 20nM panobinostat or romidepsin for 24 hours and then lysed with Triton X lysis buffer (20mM Tris-pH 7.4, 135mM NaCl, 1.5mM MgCl2, 1mM EGTA [ethyleneglycoltetraacetic acid], 10% glycerol, and 1% Triton X-100) with protease and phosphatase inhibitors. Samples were boiled for 5 minutes in loading buffer, and equal amounts of extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein was transferred to nitrocellulose membranes and blocked for 60 minutes at room temperature in PBS with 5% BSA. Membranes were incubated with polyclonal rabbit anti–pMLC2 immunoglobulin G (IgG) at a dilution of 1/1000, polyclonal rabbit anti–MLC2 IgG at a dilution of 1/1000, polyclonal rabbit anti–PAK1 IgG at a dilution of 1/1000 (Cell Signaling Technology) monoclonal mouse anti–Rac1 IgG at a dilution of 1/500 and monoclonal mouse anti–CDC42 IgG at a dilution of 1/500 (Becton Dickinson) and mouse monoclonal anti–RhoA IgG at a dilution of 1/500 and goat polyclonal anti–ROCK1 IgG at a dilution of 1/500 (Santa Cruz Biotechnology). Primary antibodies were detected with secondary antibodies conjugated with horseradish peroxidase, and filters were developed with enhanced chemiluminescence (Amersham). All experiments were performed at least 3 times.

Fluorescence microscopy

Fetal liver cell-derived megakaryocytes were isolated as described in “Proplatelet formation assay” after treatment for 48 hours with 1nM panobinostat or 10nM romidepsin. Meg-01 cells were treated with 20nM panobinostat or 20nM romidepsin for 24 hours. Cells were then centrifuged onto slides coated with poly- l -lysine, fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton X-100 for 5 minutes, and then incubated with anti-pMLC2 (Cell Signaling Technology) and anti-tubulin for 1 hour at room temperature. After washing, the cells were incubated with the appropriate secondary antibodies conjugated with Alexa Fluor 488 or 543. Cells were coverslipped with 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI) containing ProLong Gold Antifade reagent (Invitrogen) for nucleus staining. Cells were examined under a BX-51 microscope (Olympus) equipped with a 60× water objective. All experiments were performed at least 3 times.


Immunohistochemical staining was performed using the tyramide signal amplification biotin system kit (Perkin-Elmer). After de-waxing, slides underwent heat antigen retrieval by placing them in 10mM sodium citrate, pH 6.0, in a pressure cooker for 3 minutes at 125°C. After cooling and washing in PBS, slides were incubated in a humidified chamber in avidin reagent for 10 minutes, washed in PBS, incubated in biotin reagent for 10 minutes, and washed again in PBS. Free aldehydes were blocked by incubation with 0.37 g/100 mL of glycine for 5 minutes, and then blocked in TNB buffer (0.1M Tris-HCl, pH 7.5, 0.15M NaCl, and 0.5% blocking reagent [supplied in the kit]). Primary pMLC antibody was applied for 1 hour before slides were washed in TNT wash buffer (0.1M Tris-HCl, pH 7.5, 0.15M NaCl, and 0.05% Tween). Biotinylated secondary anti–rabbit IgG was applied for 30 minutes, and after washing in TNT buffer, slides were incubated with streptavidin-horseradish peroxidase for 30 minutes. After further washing, slides were incubated with biotinyl tyramide plus fluorescein amplification reagent for 6 minutes. After washing in PBS, slides were coverslipped and cells were examined under a BX-51 microscope equipped with a 20× objective. Fluorescent intensity of staining of megakaryocytes was analyzed using Metamorph v7.63 software (Molecular Devices).

Retroviral transduction and transfection

Meg-01 cells expressing constitutively active (Q61L) or dominant-negative (T17N) Rac1 constructs (kind gifts from Dr Kathy Jastrzebski, Peter MacCallum Cancer Centre, Melbourne, Australia) were engineered by retroviral transduction. Retrovirus-containing supernatant was produced by transfecting 293T packaging cells with murine stem cell virus, internal ribosome entry site Cherry-red and an amphotropic helper plasmid using standard calcium phosphate–transfection methods. Viral supernatant was used to transduce Meg-01 cells. Seventy-two hours after transduction, Cherry-red–positive cells were isolated by flow cytometry–mediated cell sorting and cultured. After expansion of cells, Western blotting for pMLC was performed as described in “Western blot analysis” on cells that were at least 90% Cherry-red positive.

DNA polymerases

DNA polymerases are critical components in PCR, since they synthesize the new complementary strands from the single-stranded DNA templates. All DNA polymerases possess 5′→ 3′ polymerase activity, which is the incorporation of nucleotides to extend primers at their 3′ ends in the 5’ to 3’ direction (Figure 2).

In the early days of PCR, the Klenow fragment of DNA polymerase I from E. coli was used to generate the new daughter strands [3]. However, this E. coli enzyme is heat-sensitive and easily destroyed at the high denaturing temperatures that precede the annealing and extension steps. Thus, the enzyme needed to be replenished at the annealing step of each cycle throughout the process.

The discovery of thermostable DNA polymerases proved to be an important advancement, opening tremendous opportunities for the improvement of PCR methods by enabling longer-term stability of the reactions. One of the best-known thermostable DNA polymerases is Taq DNA polymerase, isolated from the thermophilic bacterial species Thermus aquaticus in 1976 [5,6]. In the first report in 1988 [7], researchers demonstrated Taq DNA polymerase’s retention of activity above 75°C, making continuous cycling without manual addition of fresh enzyme possible, and thus enabling workflow automation. Furthermore, compared to E. coli DNA polymerase, Taq DNA polymerase produced longer PCR amplicons with higher sensitivity, specificity, and yield. For all the aforementioned reasons, Taq DNA polymerase was named “Molecule of the Year” by the journal Science in 1989 [8].

Figure 2. DNA polymerase extending the 3′ end of a PCR primer in the 5′ to 3′ direction.

Although Taq DNA polymerase significantly improved PCR protocols, the enzyme still presented some drawbacks. Taq DNA polymerase is relatively unstable above 90°C during denaturation of DNA strands. This is especially problematic for DNA templates with high GC content and/or strong secondary structures that require higher temperatures for separation. The enzyme also lacks proofreading activity therefore, Taq DNA polymerase can misincorporate nucleotides during amplification. Where sequence accuracy is critical, PCR amplicons with errors are not desirable for cloning and sequencing. In addition, the error-prone nature of Taq DNA polymerase contributes to its inability to amplify fragments longer than 5 kb in general. To overcome such shortcomings, better-performing DNA polymerases are continually being developed to harness the power of PCR across a variety of biological applications (learn more about DNA polymerase characteristics).

Video: Fast PCR enzyme

Achieve 4x faster DNA synthesis, anneal primers at 60°C, and load samples directly onto gels after PCR, using Invitrogen Platinum II Taq Hot-Start DNA Polymerase.


Viruses rely on their host to replicate and multiply. This is because viruses are unable to go through cell division, as they are acellular–meaning they lack the genetic information that encode the necessary tools for protein synthesis or generation of metabolic energy hence they rely on their host to replicate and multiply. Using the host cell's machinery the virus generates copies of its genome and produces new viruses for the survival of its kind and the infection of new hosts. The viral replication process varies depending on the virus's genome. [2]

Classification Edit

In 1971 David Baltimore, a Nobel Prize-winning biologist, created a system called Baltimore Classification System.According to this system, viruses are classified into seven classes based on their replication strategy: [2] [4]

  • Class I: Double-stranded DNA. Depending on the location of genome replication, this class can be subdivided into two groups: (a) viruses in which replication happens exclusively in the nucleus and is thus relatively dependent on cellular factors (b) viruses replicating in cytoplasm that are mostly independent of cellular machinery due to the fact that they have acquired all needed factors for their genome's transcription and replication. [2]
  • Class II: Single-stranded DNA. These viruses only replicate in the nucleus. A double-stranded intermediate is formed during the replication process that serves as a template for the synthesis of virus's single-stranded DNA. [2]
  • Class III: Double-stranded RNA. The viruses in this class have segmented genomes. Each segment is transcribed individually to produce a monochromatic mRNA that codes for only one protein. [2]
  • Class IV: Single-stranded RNA - Positive-sense. The genome of these viruses are positive-sense RNAs, in which the RNA is directly translated into a viral protein. These viruses can be subdivided into two groups depending on their translation process: (a) viruses with polycistronic mRNA, in which the RNA translates into multiple protein products (b) viruses with more complex transcription than the first group where either subgenomic RNAs or two rounds of translation are necessary to produce the genomic RNA. [2]
  • Class V: Single-stranded RNA - Negative-sense. All viruses in this class have negative-sense RNAs, in which the RNA complements mRNA, transcribes into a positive-sense RNA via a viral polymerase, and translates into viral protein. The viruses in this class either have segmented or unsegmented RNA either way, replication occurs within the cell's cytoplasm. [2]
  • Class VI: Single-stranded positive-sense RNA with DNA intermediate. These viruses use reverse transcriptase to convert the positive-sense RNA ( as the template) into DNA. Retroviruses are the most well-known family among this class. [2]
  • Class VII: Double-stranded DNA with RNA intermediate. The genome of these viruses are gapped, double-stranded, and subsequently become filled to form cccDNA (covalently closed circular DNA). This group also use the reverse transcription during the process of maturation. [2]

Replication cycle Edit

Regardless of the differences among viral species, they all share six basic replication stages: [2] [5]

Attachment is the cycle's starting point and consists of specific bindings between anti-receptors (or virus-attachment proteins) and cellular receptors molecules such as (glyco)proteins. The host range of a virus is determined by the specificity of the binding. The attachment causes the viral protein to change its configuration and thus fuse with the host's cell membrane thereby enabling the virus to enter the cell. [6]

Penetration of virus happens either through membrane fusion or receptor-mediated endocytosis and leads to viral entry. Due to their rigid cellulose-made

(chitin in case of fungal cells) cell walls, plants and fungal cells get infected differently than animal cells. Often, a cell wall trauma is required for the virus to enter the cell. [6]

Uncoating is the removal of viral capsid, which makes the viral nucleic acids available for transcription. The capsid could have been degraded by either host or viral enzymes, releasing the viral genome into the cell. [6]

Replication is the multiplication of virus's genetic material. The process includes the transcription of mRNA, synthesis, and assembly of viral proteins and is regulated by protein expression. [6]

Assembly process involves putting together and modifying newly made viral nucleic acids and structural proteins to form the virus's nucleocapsid. [2] [7]

Release of viruses could be done by two different mechanisms depending on the type of virus. Lytic viruses burst the cell's membrane or wall through a process called lysis in order to release themselves. On the other hand, enveloped viruses become released by a process called budding in which a virus obtain its lipid membrane as it buds out of the cell through membrane or intracellular vesicle. Both lysis and budding processes are highly damaging to the cell, with the exception of retroviruses, and often result in the cell's death. [2] [6]

Pathogenicity is the ability of one organism to cause disease in another. There is a specialized field of study in virology called viral pathogenesis in which it studies how viruses infect their hosts at the molecular and cellular level. [2]

In order for the viral disease to develop several steps need to be taken. First, the virus has to enter the body and implant itself into a tissue ( e.g. respiratory tissue). Second, the virus has to reproduce extracellular after invading in order to make ample copies of itself. Third, the synthesized viruses must spread throughout the body via circulatory systems or nerve cells. [8]

With regards to viral diseases it is essential to look at two aspects: (a) the direct effect of virus replication (b) the body's responses to infection. The course and severity of all viral infections are determined by the dynamic between the virus and the host. Common symptoms of virus infections are fever, body aches, inflammation, and skin rashes. Most of these symptoms are caused by our immune system's response to infection and are not the direct effect of viral replication. [2]

Cytopathis effects of virus Edit

Often it is possible to recognize virus-infected cells through a number of common phenotypic changes referred to as cytopathis effects. These changes include: [2]

  • Altered shape: The shape of adherent cells– cells that attach themselves to other cells or artificial substrates— may change from flat to round. In addition, the cell's extensions resembling tendrils – involved in cell's mobility and attachments—are withdrawn into the cell. [2]
  • Detachment from the substrate: Happens as a result of adherent cells' damage. The cell damage is due to partial degradation of cytoskeleton. [2]
  • Lysis: In this case the entire cell breaks down because of the absorption of extracellular fluid and swelling. Not all viruses cause lysis. [2] : The adjacent cells' membranes fuse together and produce a mass called syncytium, in which the cytoplasm contains more than one nucleus and appears as a giant cell. These cells have a short lifespan compared to other cells. [2]
  • Increase in Membrane permeability: Viruses can increase the membrane permeability which allows many extracellular ions( e.g. iodine and sodium ions) to enter the cell. [2]

There are five different types of viral infections: [2]

    : This kind of infection occurs when a virus successfully invades a host cell but is unable to complete its full replication cycle and produce more infectious viruses. [2]
  • Acute infection: Many common viral infections fallow this pattern. Acute infections are brief since they are often completely eliminated by the immune system. Acute infection is frequently associated with epidemics since most of virus replication happens before the onset of symptoms. [2]
  • Chronic infection: These infections have a prolonged course and are hard to eliminate since the virus stays in the host for a significant period. [2]
  • Persistent infection: There is a delicate balance between the host and the virus in this pattern. The virus adjusts its replication and pathogenecitiy levels to keep the host alive for its own benefit. While it is possible for the virus to live and replicate inside the host for its entire lifetime, oftentimes the host eventually eliminates the virus. [2]
  • Latent infection: Being the ultimate infection, the latent virus infections tend to exist inside the host for its entire lifetime. A well-known example of such infection is the herpes simplex virus in humans. This virus is able to stop its replication and restrict its gene expression in order to stop the recognition of the infected cell by the host's immune system. [2]

Prevention and treatment Edit

Viral vaccines contain inactivated viruses which have lost their ability to replicate. These vaccines can prevent or lower the intensity of viral illness. Developing vaccines against smallpox, polio, and hepatitis B over the past 50 years has had a significant impact on world health and thus on global population. Nevertheless, there have been ongoing viral outbreaks ( such as the Ebola and Zika viruses) in the past few years affecting millions of people all around the globe. [9] Therefore, more advanced understanding of molecular virology and viruses are needed for the development of new vaccines and the control of ongoing/future viral outbreaks. [2]

An alternative way to treat viral infections would be antiviral drugs in which the drug blocks the virus's replication cycle. The specificity of an antiviral drug is the key to its success. These drugs are toxic to both the virus and the host but in order to minimize their damage they are developed in such a way as to be more toxic to the virus than to the host. The efficiency of an antiviral drug is measured by the chemotherapeutic index, given by: [2]


Molecular mechanics is one aspect of molecular modelling, as it involves the use of classical mechanics (Newtonian mechanics) to describe the physical basis behind the models. Molecular models typically describe atoms (nucleus and electrons collectively) as point charges with an associated mass. The interactions between neighbouring atoms are described by spring-like interactions (representing chemical bonds) and Van der Waals forces. The Lennard-Jones potential is commonly used to describe the latter. The electrostatic interactions are computed based on Coulomb's law. Atoms are assigned coordinates in Cartesian space or in internal coordinates, and can also be assigned velocities in dynamical simulations. The atomic velocities are related to the temperature of the system, a macroscopic quantity. The collective mathematical expression is termed a potential function and is related to the system internal energy (U), a thermodynamic quantity equal to the sum of potential and kinetic energies. Methods which minimize the potential energy are termed energy minimization methods (e.g., steepest descent and conjugate gradient), while methods that model the behaviour of the system with propagation of time are termed molecular dynamics.

This function, referred to as a potential function, computes the molecular potential energy as a sum of energy terms that describe the deviation of bond lengths, bond angles and torsion angles away from equilibrium values, plus terms for non-bonded pairs of atoms describing van der Waals and electrostatic interactions. The set of parameters consisting of equilibrium bond lengths, bond angles, partial charge values, force constants and van der Waals parameters are collectively termed a force field. Different implementations of molecular mechanics use different mathematical expressions and different parameters for the potential function. [2] The common force fields in use today have been developed by using chemical theory, experimental reference data, and high level quantum calculations. The method, termed energy minimization, is used to find positions of zero gradient for all atoms, in other words, a local energy minimum. Lower energy states are more stable and are commonly investigated because of their role in chemical and biological processes. A molecular dynamics simulation, on the other hand, computes the behaviour of a system as a function of time. It involves solving Newton's laws of motion, principally the second law, F = m a =mmathbf > . Integration of Newton's laws of motion, using different integration algorithms, leads to atomic trajectories in space and time. The force on an atom is defined as the negative gradient of the potential energy function. The energy minimization method is useful to obtain a static picture for comparing between states of similar systems, while molecular dynamics provides information about the dynamic processes with the intrinsic inclusion of temperature effects.

Molecules can be modelled either in vacuum, or in the presence of a solvent such as water. Simulations of systems in vacuum are referred to as gas-phase simulations, while those that include the presence of solvent molecules are referred to as explicit solvent simulations. In another type of simulation, the effect of solvent is estimated using an empirical mathematical expression these are termed implicit solvation simulations.

Coordinate representations Edit

Most force fields are distance-dependent, making the most convenient expression for these Cartesian coordinates. Yet the comparatively rigid nature of bonds which occur between specific atoms, and in essence, defines what is meant by the designation molecule, make an internal coordinate system the most logical representation. In some fields the IC representation (bond length, angle between bonds, and twist angle of the bond as shown in the figure) is termed the Z-matrix or torsion angle representation. Unfortunately, continuous motions in Cartesian space often require discontinuous angular branches in internal coordinates, making it relatively hard to work with force fields in the internal coordinate representation, and conversely a simple displacement of an atom in Cartesian space may not be a straight line trajectory due to the prohibitions of the interconnected bonds. Thus, it is very common for computational optimizing programs to flip back and forth between representations during their iterations. This can dominate the calculation time of the potential itself and in long chain molecules introduce cumulative numerical inaccuracy. While all conversion algorithms produce mathematically identical results, they differ in speed and numerical accuracy. [3] Currently, the fastest and most accurate torsion to Cartesian conversion is the Natural Extension Reference Frame (NERF) method. [3]

Molecular modelling methods are now used routinely to investigate the structure, dynamics, surface properties, and thermodynamics of inorganic, biological, and polymeric systems. The types of biological activity that have been investigated using molecular modelling include protein folding, enzyme catalysis, protein stability, conformational changes associated with biomolecular function, and molecular recognition of proteins, DNA, and membrane complexes. [4]


The field of molecular biology grew in the late twentieth century, as did its clinical application. In 1980, Yuet Wai Kan et al. suggested a prenatal genetic test for Thalassemia that did not rely upon DNA sequencing—then in its infancy—but on restriction enzymes that cut DNA where they recognised specific short sequences, creating different lengths of DNA strand depending on which allele (genetic variation) the fetus possessed. [6] In the 1980s, the phrase was used in the names of companies such as Molecular Diagnostics Incorporated [7] and Bethseda Research Laboraties Molecular Diagnostics. [8] [9]

During the 1990s, the identification of newly discovered genes and new techniques for DNA sequencing led to the appearance of a distinct field of molecular and genomic laboratory medicine in 1995, the Association for Molecular Pathology (AMP) was formed to give it structure. In 1999, the AMP co-founded The Journal of Medical Diagnostics. [10] Informa Healthcare launched Expert Reviews in Medical Diagnostics in 2001. [1] From 2002 onwards, the HapMap Project aggregated information on the one-letter genetic differences that recur in the human population—the single nucleotide polymorphisms—and their relationship with disease. [2] ( ch 37 ) In 2012, molecular diagnostic techniques for Thalassemia use genetic hybridization tests to identify the specific single nucleotide polymorphism causing an individual's disease. [11]

As the commercial application of molecular diagnostics has become more important, so has the debate about patenting of the genetic discoveries at its heart. In 1998, the European Union's Directive 98/44/ECclarified that patents on DNA sequences were allowable. [12] In 2010 in the US, AMP sued Myriad Genetics to challenge the latter's patents regarding two genes, BRCA1, BRCA2, which are associated with breast cancer. In 2013, the U.S. Supreme Court partially agreed, ruling that a naturally occurring gene sequence could not be patented. [13] [14]

Development from research tools Edit

The industrialisation of molecular biology assay tools has made it practical to use them in clinics. [2] ( foreword ) Miniaturisation into a single handheld device can bring medical diagnostics into the clinic and into the office or home. [2] ( foreword ) The clinical laboratory requires high standards of reliability diagnostics may require accreditation or fall under medical device regulations. [15] As of 2011 [update] , some US clinical laboratories nevertheless used assays sold for "research use only". [16]

Laboratory processes need to adhere to regulations, such as the Clinical Laboratory Improvement Amendments, Health Insurance Portability and Accountability Act, Good Laboratory Practice, and Food and Drug Administration specifications in the United States. Laboratory Information Management Systems help by tracking these processes. [17] Regulation applies to both staff and supplies. As of 2012 [update] , twelve US states require molecular pathologists to be licensed several boards such as the American Board of Medical Genetics and the American Board of Pathology certify technologists, supervisors, and laboratory directors. [18]

Automation and sample barcoding maximise throughput and reduce the possibility of error or contamination during manual handling and results reporting. Single devices to do the assay from beginning to end are now available. [15]

Assays Edit

Molecular diagnostics uses in vitro biological assays such as PCR-ELISA or Fluorescence in situ hybridization. [19] [20] The assay detects a molecule, often in low concentrations, that is a marker of disease or risk in a sample taken from a patient. Preservation of the sample before analysis is critical. Manual handling should be minimised. [21] The fragile RNA molecule poses certain challenges. As part of the cellular process of expressing genes as proteins, it offers a measure of gene expression but it is vulnerable to hydrolysis and breakdown by ever-present RNAse enzymes. Samples can be snap-frozen in liquid nitrogen or incubated in preservation agents. [2] ( ch 39 )

Because molecular diagnostics methods can detect sensitive markers, these tests are less intrusive than a traditional biopsy. For example, because cell-free nucleic acids exist in human plasma, a simple blood sample can be enough to sample genetic information from tumours, transplants or an unborn fetus. [2] ( ch 45 ) Many, but not all, molecular diagnostics methods based on nucleic acids detection use polymerase chain reaction (PCR) to vastly increase the number of nucleic acid molecules, thereby amplifying the target sequence(s) in the patient sample. [2] ( foreword ) PCR is a method that a template DNA is amplified using synthetic primers, a DNA polymerase, and dNTPs. The mixture is cycled between at least 2 temperatures: a high temperature for denaturing double-stranded DNA into single-stranded molecules and a low temperature for the primer to hybridize to the template and for the polymerase to extend the primer. Each temperature cycle theoretically doubles the quantity of target sequence. Detection of sequence variations using PCR typically involves the design and use oligonucleotide reagents that amplify the variant of interest more efficiently than wildtype sequence. PCR is currently the most widely used method for detection of DNA sequences. [22] The detection of the marker might use real time PCR, direct sequencing, [2] ( ch 17 ) microarray chips—prefabricated chips that test many markers at once, [2] ( ch 24 ) or MALDI-TOF [23] The same principle applies to the proteome and the genome. High-throughput protein arrays can use complementary DNA or antibodies to bind and hence can detect many different proteins in parallel. [24] Molecular diagnostic tests vary widely in sensitivity, turn around time, cost, coverage and regulatory approval. They also vary in the level of validation applied in the laboratories using them. Hence, robust local validation in accordance with the regulatory requirements and use of appropriate controls is required especially where the result may be used to inform a patient treatment decision. [25]

Prenatal Edit

Conventional prenatal tests for chromosomal abnormalities such as Down Syndrome rely on analysing the number and appearance of the chromosomes—the karyotype. Molecular diagnostics tests such as microarray comparative genomic hybridisation test a sample of DNA instead, and because of cell-free DNA in plasma, could be less invasive, but as of 2013 it is still an adjunct to the conventional tests. [26]

Treatment Edit

Some of a patient's single nucleotide polymorphisms—slight differences in their DNA—can help predict how quickly they will metabolise particular drugs this is called pharmacogenomics. [27] For example, the enzyme CYP2C19 metabolises several drugs, such as the anti-clotting agent Clopidogrel, into their active forms. Some patients possess polymorphisms in specific places on the 2C19 gene that make poor metabolisers of those drugs physicians can test for these polymorphisms and find out whether the drugs will be fully effective for that patient. [28] Advances in molecular biology have helped show that some syndromes that were previously classed as a single disease are actually multiple subtypes with entirely different causes and treatments. Molecular diagnostics can help diagnose the subtype—for example of infections and cancers—or the genetic analysis of a disease with an inherited component, such as Silver-Russell syndrome. [1] [29]

Infectious disease Edit

Molecular diagnostics are used to identify infectious diseases such as chlamydia, [30] influenza virus [31] and tuberculosis [32] or specific strains such as H1N1 virus [33] or SARS-CoV-2. [34] Genetic identification can be swift for example a loop-mediated isothermal amplification test diagnoses the malaria parasite and is rugged enough for developing countries. [35] But despite these advances in genome analysis, in 2013 infections are still more often identified by other means—their proteome, bacteriophage, or chromatographic profile. [36] Molecular diagnostics are also used to understand the specific strain of the pathogen—for example by detecting which drug resistance genes it possesses—and hence which therapies to avoid. [36] In addition, assays based on metagenomic next generation sequencing can be implemented to identify pathogenic organisms without bias. [37]

Disease risk management Edit

A patient's genome may include an inherited or random mutation which affects the probability of developing a disease in the future. [27] For example, Lynch syndrome is a genetic disease that predisposes patients to colorectal and other cancers early detection can lead to close monitoring that improves the patient's chances of a good outcome. [38] Cardiovascular risk is indicated by biological markers and screening can measure the risk that a child will be born with a genetic disease such as Cystic fibrosis. [39] Genetic testing is ethically complex: patients may not want the stress of knowing their risk. [40] In countries without universal healthcare, a known risk may raise insurance premiums. [41]

Cancer Edit

Cancer is a change in the cellular processes that cause a tumour to grow out of control. [27] Cancerous cells sometimes have mutations in oncogenes, such as KRAS and CTNNB1 (β-catenin). [42] Analysing the molecular signature of cancerous cells—the DNA and its levels of expression via messenger RNA—enables physicians to characterise the cancer and to choose the best therapy for their patients. [27] As of 2010, assays that incorporate an array of antibodies against specific protein marker molecules are an emerging technology there are hopes for these multiplex assays that could measure many markers at once. [43] Other potential future biomarkers include micro RNA molecules, which cancerous cells express more of than healthy ones. [44]

Cancer is a disease with excessive molecular causes and constant evolution. There’s also heterogeneity of disease even in an individual. Molecular studies of cancer have proved the significance of driver mutations in the growth and metastasis of tumors. [45] Many technologies for detection of sequence variations have been developed for cancer research. These technologies generally can be grouped into three approaches: polymerase chain reaction (PCR), hybridization, and next-generation sequencing (NGS). [22] Currently, a lot of PCR and hybridization assays have been approved by FDA as in vitro diagnostics. [46] NGS assays, however, are still at an early stage in clinical diagnostics. [47]

To do the molecular diagnostic test for cancer, one of the significant issue is the DNA sequence variation detection. Tumor biopsy samples used for diagnostics always contain as little as 5% of the target variant as compared to wildtype sequence. Also, for noninvasive applications from peripheral blood or urine, the DNA test must be specific enough to detect mutations at variant allele frequencies of less than 0.1%. [22]

Currently, by optimizing the traditional PCR, there’s a new invention, amplification-refractory mutation system (ARMS) is a method for detecting DNA sequence variants in cancer. The principle behind ARMS is that the enzymatic extension activity of DNA polymerases is highly sensitive to mismatches near the 3′ end of primer. [22] Many different companies have developed diagnostics tests based on ARMS PCR primers. For instance, Qiagen therascreen, [48] Roche cobas [49] and Biomerieux THxID [50] have developed FDA approved PCR tests for detecting lung, colon cancer and metastatic melanoma mutations in the KRAS, EGFR and BRAF genes. Their IVD kits were basically validated on genomic DNA extracted from FFPE tissue.

There’s also microarrays that utilize hybridization mechanism to do diagnostics of cancer. More than a million of different probes can be synthesized on an array with Affymetrix's Genechip technology with a detection limit of one to ten copies of mRNA per well. [22] Optimized microarrays are typically considered to produce repeatable relative quantitation of different targets. [51] Currently, FDA have already approved a number of diagnostics assays utilizing microarrays: Agendia's MammaPrint assays can inform the breast cancer recurrence risk by profiling the expression of 70 genes related to breast cancer [52] Autogenomics INFNITI CYP2C19 assay can profile genetic polymorphisms, whose impacts on therapeutic response to antidepressants are great [53] and Affymetrix's CytoScan Dx can evaluate intellectual disabilities and congenital disorders by analyzing chromosomal mutation. [54]

In the future, the diagnostic tools for cancer will likely to focus on the Next Generation Sequencing (NGS). By utilizing DNA and RNA sequencing to do cancer diagnostics, technology in the field of molecular diagnostics tools will develop better. Although NGS throughput and price have dramatically been reduced over the past 10 years by roughly 100-fold, we remain at least 6 orders of magnitude away from performing deep sequencing at a whole genome level. [55] Currently, Ion Torrent developed some NGS panels based on translational AmpliSeq, for example, the Oncomine Comprehensive Assay. [56] They are focusing on utilizing deep sequencing of cancer-related genes to detect rare sequence variants. The advantages of NGS are reduced reagent consumption and highly controlled microenvironments, specially when it is combined with microfluidic devices. [57]

Molecular diagnostics tool can be used for cancer risk assessment. For example, the BRCA1/2 test by Myriad Genetics assesses women for lifetime risk of breast cancer. [22] Also, some cancers are not always employed with clear symptoms. It is useful to analyze people when they do not show obvious symptoms and thus can detect cancer at early stages. For example, the ColoGuard test may be used to screen people over 55 years old for colorectal cancer. [58] Cancer is a longtime-scale disease with various progression steps, molecular diagnostics tools can be used for prognosis of cancer progression. For example, the OncoType Dx test by Genomic Health can estimate risk of breast cancer. Their technology can inform patients to seek chemotherapy when necessary by examining the RNA expression levels in breast cancer biopsy tissue. [59]

With rising government support in DNA molecular diagnostics, it is expected that an increasing number of clinical DNA detection assays for cancers will become available soon. Currently, research in cancer diagnostics are developing fast with goals for lower cost, less time consumption and simpler methods for doctors and patients.


In the present study, we have characterized the signal transduction system regulating the serum deprivation-dependent stimulation of the NHE1 in the advanced breast carcinoma cell lines MDA-MB-435 and MDA-MB-231 and in the less invasive MCF-7 cell line derived from a primary tumor. We describe a novel signal transduction module localized in dominant leading edge pseudopodia of breast cancer cells that, during serum deprivation, integrates PKA, RhoA, p160ROCK, and p38α into a phosphorylation-triggered signal cascade hierarchy that controls tumor cell NHE1 activity and invasive capacity. PKA acts through phosphorylation of RhoA on serine 188 to release the suppression of NHE1 activity by the RhoA/p160ROCK/p38α pathway (Figure 8).

Many studies have underlined the importance of PKA as a gating element in a number of different signaling systems and an important example of this is the gating of integrin-stimulated invasion in human breast (O'Connor et al., 1998), colon (O'Connor et al., 2000) cancer cells and in luteinizing hormone regulation of human endometrial cancer cells (Dabizzi et al., 2003), demonstrating a key regulatory role for PKA in the cancer invasive process. RhoA is known to be overexpressed in breast cancer cells and to play a role in increased invasion (Lin and van Golen, 2004), and one PKA gating mechanism in normal cells is the PKA phosphorylation of RhoA on Ser188, which leads to its increased removal from the plasma membrane (Lang et al., 1996 Forget et al., 2002) and to a decreased association with its downstream effector p160ROCK (Laudanna et al., 1997 Busca et al., 1998). Whereas PKA and RhoA have been shown to have opposite effects on cancer cell migration (O'Connor et al., 2000), our results demonstrate for the first time in cancer cells the importance of direct PKA-dependent phosphorylation and inhibition of RhoA in the invasive process.

Figure 7. Role of PKA/RhoA/ROCK/p38 signal module in serum deprivation-dependent regulation of NHE1 and cell shape in MCF-7 (left) and MDA-MB-231 (right) breast cancer cell lines. (A) PKA-dependent potentiation of serum deprivation-induced NHE1 activity is blocked by inhibiting p38 and role of phosphorylation at serine 188 in stimulation of NHE1 activity by serum deprivation in both MCF-7 and MDA-MB-231 cell lines. Experiments were conducted as described in Figure 3 and 4 legends. Data are the mean ± SE of between nine and 12 observations for each condition. ††† p < 0.001 and †† p < 0.01 compared with nondeprived control, whereas **p < 0.001 and **p < 0.01 compared with deprived control. (B) Epifluorescence images of colabeled total RhoA and phospho-RhoA cells in MCF-7 and MDA-MB-231 cells treated for 24 h in serum replete (NonDeprived) or deprived medium. Arrowheads indicate pseudopodial-located phospho RhoA and RhoA. (C) Serum deprivation induces PKA-dependent inhibition of RhoA activity in the pseudopodia of both MCF-7 and MDA-MB-231 cells. Experiments were conducted as described in Figure 6B legend, and the number of experiments is indicated in their respective bars. ††† p < 0.001 and †† p < 0.01 compared with nondeprived control cells and ***p < 0.001 compared with main body of deprived cells. n.s., not significant.

The orchestration of complex cellular events, such as motility or invasion, involves the assembly of multimolecular complexes or modules at the cellular site of action (Hancock and Moon, 2000). It is now well established that tumor cells have acquired altered morphological characteristics to facilitate their increased invasive ability (Lagana et al., 2000) and establishment of the directed cell polarity involved in invasion requires dynamic remodeling of the cytoskeleton and sorting of proteins to the leading-edge pseudopodial compartment. Serum deprivation provoked an extension of the leading edge pseudopodia, a compartment that has been previously identified to be involved in cancer cell invasion (Liotta and Clair, 2000 Taguchi et al., 2000). This serum deprivation-dependent extension was accompanied by a redistribution of NHE1, RhoA and an increase in phospho-RhoA along the trunk and, especially strong, at the distal tip (Figures 6A and 7C). Furthermore, we observe here that in the cancer cell serum deprivation provokes an inhibition of RhoA activity preferentially in the leading edge pseudopodia that is totally dependent on its phosphorylation by PKA (Figures 6, B and C, and 7D), whereas in normal, nontumor cells (MCF-10A) the RhoA response to serum deprivation occurs evenly throughout the cell and was independent of PKA activity (Supplemental Figure 3). Together, the data presented here support the idea that in breast cancer cells serum deprivation locally activates a PKA controlled signaling module organized within an invasion-specific cell structure that leads to a more efficient invasive behavior. We hypothesize that the protein clustering and inhibition of RhoA in the leading edge pseudopodia is necessary for its formation and for invasion (Figure 8). This association of increased motility with an inhibition of RhoA activity is similar to that recently reported for the tight junction protein NZO-3 in controlling renal cell motility (Wittchen et al., 2003), inhibition of motility, and invasion by dihydromotuporamine C (McHardy et al., 2004), tenascin-induced colon cancer cell invasion (De Wever et al., 2004) or by the degradation of RhoA by Smurf1 in controlling transformed Mv1Lu and HEK293T cell motility (Wang et al., 2003), suggesting a common mechanism. This last work observed RhoA degradation primarily in the cell protrusions supporting our present results concerning the loss of RhoA activity, although the underlying mechanism is different. Interestingly, localized activation of a RhoA/ROCK/p38MAPK signaling module to the pseudopodial domain of invasive Moloney sarcoma virus-transformed epithelial cells (MSV-MDCK-INV) regulates NHE1-dependent pseudopodial extension and motility (Jia, Noël, and Nabi, personal communication). Together, these data suggest that the RhoA/ROCK/p38MAPK signal module may be common to transformed/cancer cell regulation of invasive potential by various extracellular signals. Intriguingly, a similar inhibition of the RhoA/ROCK pathway has been reported in controlling the process of neurite outgrowth (Busca et al., 1998 Luo, 2000 Scaife et al., 2003 Yuan et al., 2003 Kishida et al., 2004), and in the latter article, it was serum deprivation that induced neuroblastoma neurite outgrowth dependent on the inhibition of RhoA. These data suggest that neurite outgrowth has profound similarities to the tumor-invasive process.

Figure 8. Model of serum deprivation-induced signal transduction module that activates the NHE1 and promotes invasion in breast cancer cells. A reduced level of serum in the tumor metabolic microenvironment increases the activity of PKA and induces a shift from a signal transduction module that excludes PKA phosphorylation of RhoA to another in which this phosphorylation is possible. This phosphorylation inhibits the activity of RhoA, resulting in the subsequent inhibition of p160ROCK and p38 MAP Kinase. The down-regulation of this NHE1 repressor signaling module results in the stimulation of NHE1 activity, the formation of a leading edge pseudopodia and subsequent invasive capacity.

The data presented in this article are important in the context of the critical role of subcellular localization and timing of PKA signaling in determining specificity of downstream regulatory processes (Zaccolo et al., 2002 Tasken and Aandahl, 2004). This difference in PKA-dependent signaling is of extreme interest clinically because studies have demonstrated both positive as well as negative cAMP/PKA modulation of tumor cell proliferation and apoptotic response, thereby rendering a unified therapeutic approach difficult (Lerner et al., 2000 Skalhegg and Tasken, 2000). A possible explanation for these seemingly contradictory reports concerning PKA regulation may be that the pleiotrophic action of PKA in cell function is based on the fine control of its spatiotemporal regulation. In this context, we recently demonstrated that treatment of these same breast cancer cells with the antineoplastic agent paclitaxel induces apoptosis via a direct PKA-induced stimulation of p38 MAP kinase, resulting in inhibition of NHE1 and subsequent induction of apoptosis (Reshkin et al., 2003). This could activate an alternative PKA-dependent signal module that differently regulates p38. The relative relationship between the last two downstream components of each signaling module remained the same: an increase in p38 activity inhibited the NHE1, whereas a decrease in p38 activity stimulated the NHE1.

Our results are consistent with a model in which the local cAMP pulse initiates a shift from a signal transduction module excluding PKA phosphorylation of RhoA to another in which this phosphorylation is possible. We suggest that this could be due to an altered expression/distribution of a PKA anchoring and/or a scaffolding protein. These protein modules are organized into physically and functionally distinct units that can reorganize when cell conditions change (Yaffe and Cantley, 1999). Ezrin is an actin-binding and PKA-anchoring protein that has been shown to also bind to NHE1 as a part of NHE1-dependent regulation of actin cytoskeletal organization and motility (Denker et al., 2000). RhoA through p160ROCK has been demonstrated to regulate both NHE1 activity (Tominaga et al., 1998) and ezrin phosphorylation (Shaw et al., 1998) and location (Kotani et al., 1997). The ezrin binding protein 50 (or NHERF1) is a scaffolding protein known to link ezrin to various proteins and thereby regulate their PKA phosphorylation state (Voltz et al., 2001). Together, these studies suggest that ezrin and NHERF could play important roles in the assembly and regulation of the serum deprivation-dependent signaling module described in the present study.

In conclusion, our results demonstrate that serum deprivation up-regulates the activity of tumor cell NHE1 via a novel PKA-gated signal transduction module that is locally activated in leading edge pseudopodia. These data demonstrate that tumor cells have developed different regulatory patterns that allow them to adapt to and take advantage of the specialized conditions of their microenvironment to maximize their neoplastic potential. The preferential activation of this invasion-specific signal module in the cellular structure specific for invasion, the pseudopodia, contributes further to the identification of the biochemical/structure-determined complexes fundamental for the digestion and infiltration of surrounding tissues by the cancer cell.

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