Supplementary Online Materials for:

Formation of regulatory patterns during signal propagation in a mammalian cellular network

Avi Maayan, Sherry L. Jenkins, Susana Neves, Anthony Hasseldine, Elizabeth Grace, Benjamin
Dubin-Thaler, Narat J. Eungdamrong, Gehzi Weng, Prahlad Ram, J. Jeremy Rice,
Aaron Kershenbaum, Gustavo A. Stolovitzky, Robert D. Blitzer, and Ravi Iyengar

Contents
Methods
Supporting text
Supporting Figures S1- S11
Supporting Table S1
Supporting references
Author contributions
Table of supporting external files: text files, source code segments, movies and spreadsheets

Methods
Constructing the network:
We used published research literature to identify the key components of signaling pathways
and cellular machines, and their binary interactions. Most components (~80%) have been described
in hippocampal neurons or related neuronal cells. Other components are from other cells, but are
included because they are key components in processes known to occur in hippocampal neurons,
such as translation. We then established that these interactions were both direct and functionally
relevant. All of the connections were individually verified by at least one of the authors of this paper
by reading the relevant primary paper(s). We developed a system made of 545 components (nodes)
and 1259 links (connections). We used arbitrary but consistent rules to sort components into various
groups. For instance, transcription factors are considered a as part of the transcriptional machinery,
although it may also be equally valid to consider them as the most downstream component of the
central signaling network. Similarly the AMPA receptor-channel (AMPAR) is considered part of the
ion channels in the electrical response system since its activity is essential to defining the
postsynaptic response, although it binds to and is activated by glutamate, and hence can be also
considered a ligand gated receptor-channel in the plasma membrane.
The links were specified by two criteria: function and biochemical mechanism. Three types
of functional links were specified. This follows the rules used for representation of pathways in

Sciences STKE (S1). Links may be activating, inhibitory or neutral. Neutral links do not specify
directionality between components, and are mostly used to represent scaffolding and anchoring
undirected or bidirectional interactions. The biochemical specification includes defining the
reactions as non-covalent binding interactions or enzymatic reactions. Within the enzymatic
category, reactions were further specified as phosphorylation, dephosphorylation, hydrolysis, etc.
These two criteria for specification are independent and were defined for all interactions. For the
analyses in this study we only used the functional criteria: activating, inhibitory or neutral
specifications.
We chose papers that demonstrated direct interactions that were supported by either
biochemical or physiological effects of the interactions. From these papers we identified the
components and interactions that make up the system we analyzed. During this specification process
we did not consider whether these interactions would come together to form higher order
organizational units. Each component and interaction was validated by a reference from the primary
literature (1202 papers were used). A list of authors who read the papers to validate the components
and interactions is provided under authors contributions.
Storage of the network data:
The data describing the components (nodes) and their interactions (links) that compose the
network are stored in a text file, see Text file S1, using the flat file *sig format described below.
The *sig flat text file format:
Source Name: cellular component that is affecting a target component
Source Human Accession: Swiss-Prot accession if available
Source Mouse Accession: Swiss-Prot accession if available
Source Type: the type of molecule classification this component
Source Location: cellular localization of the component
Target Name: generalized cellular component that is affected by the source component
Target Human Accession: Swiss-Prot accession if available
Target Mouse Accession: Swiss-Prot accession if available
Target Type: the type of molecule classification for this component
Target Location: generalized cellular localization of the component
Effect: activation (+), inhibition (-), or neutral (0)
Type of Interaction: type of chemical interaction directly linking the two components
PubMed ID: PubMed database accession number

Sorting cellular components (nodes) into cellular machines:

Components (nodes) were separated into functional machines based on their molecule type and
location attributes using Code segment S7.
Visualization of the entire network:
The diagram of the network in figure S1 was created with Code segment S8 that creates Text file
S2. This text file can be loaded into the Pajek software for visualization (S2).
Subnetworks obtained by signal propagation from ligands:
To generate these subnetworks we used a depth-first search algorithm expanding the search solely in
the downstream direction, to confirm with biologically specified directional transfer of information.
2

For example, when a Gs protein is activated by an upstream ligand-receptor interaction it connects

only to the downstream component adenylyl cyclase. The subnetworks do not include another Gs
coupled receptor unless that receptor also interacts with the specific ligand.
The recursive algorithm (using depth-first search) is listed as Code segment S9. It was used to
create 15 subnetworks for each node of the type Ligand by changing the number of steps from the
ligand. This concept is termed breadth-first search and is illustrated in Movie S20. The subnetworks
were then analyzed by computing the number of nodes, links, clustering, average path length and
motifs.
Visualization of the resulting data for connections per step for all ligands, Spreadsheet S23, was
created with MatlabTM, Natick, MA (S3).
Three sets of subnetworks induced by the nodes: GLUTAMATE, NE and BDNF were analyzed for
number of nodes and links, clustering, average path-length and motifs with Code segment S10.
These results, Spreadsheet S24, were visualized with ExcelTM (S4).
Overall statistics of the network:
The connectivity distribution data, Spreadsheet S25, was plotted with MatlabTM, Natick, MA (S3).
Characteristic path-length measure was implemented by using Floyds algorithm (S5), with Code
segment S11.
Clustering Coefficient was implemented with custom code based on the concept developed by Watts
and Strogatz (S6), using Code segment S12.
Grid Coefficient was implemented with custom code based on the concept developed by Caldarelli
et al. (S7), using Code segment S13.
The results, Spreadsheet S26, were compared to the same parameters computed for 100 shuffled
networks of one island that maintain the same connectivity distribution using an algorithm
developed by Milo et al. (S8), using Code segment S14.
Network Motifs:
Counting motifs in the network was accomplished by using the MFinder program developed by
Kashtan et al. (S9). Motifs of size 3, 4, 5 and 6 were visualized using VisioTM (S10).
See Table S29 of the complete results for the motifs of sizes 3 and 4.
Output files in text format from the MFinder program:
Motifs size 3: Text file S3.
Motifs size 4: Text file S4.
Motifs size 5: Text file S5.
Motifs size 6: Text file S6.
Our method for motif search:
In our detailed analysis of subnetworks, we only considered closed loop circular motifs. We
searched for these motifs using a depth-first search algorithm (Code segments S17 through S19).
An example of counting motifs in a toy network is given below:

The above toy network contain: 1 positive feedback loop of size 3, 1 negative feedback loop of size
3 and 1 negative feed-forward motif of size 4. Nodes C and D contribute to 3 motifs whereas nodes
B and C contribute to 2 motifs. This is slightly different from the method used by Kashtan et al. (S9)
because, with their method, A, B, C, and D will not be considered a feedforward loop due to the link
between A and D.
Positive vs. negative feedback and feedforward loops:
Positive feedback loops motifs are defined as loops where all the links in the loop are positive
(activating), or there are an even number of negative (inhibitory) links. Negative feedback loops are
loops where there is an odd number of negative links. Feedforward loops have two arms, each
starting at a source node and merging into a target (sink) node. To determine if a feedforward loop is
positive or negative, each arm was evaluated separately. Both arms must be positive for the
feedforward motifs to be positive. An arm that has all links positive or even number of negative
links is considered positive; whereas, an arm with an odd number of negative links is considered
negative.
Subnetworks from source to target nodes:
Series of subnetworks from specific source nodes (generally ligands) to specific targets
(components within machines such as channels or transcription factors), with limited path lengths,
were created using the following code: Code segment S15. The concept is illustrated in Movie S21.
Shuffled networks, where only links that do not involved the source nodes and target nodes, were
created for statistical control. In the control subnetworks, we preserved the biologically specified
connectivity and directionality between ligands and their receptors and from the immediate
upstream components to the AMPA channel and CREB. For all other connections we randomly
swapped the direction of the connection while preserving the overall connectivity structure of the
network. Curve fitting of the data, Spreadsheet S27, was done with ExcelTM (S4).
Subnets based on nodal connectivity:
Series of sub-networks created based on nodal connectivity were created using Code segment S16.
The concept is illustrated in Movie S22. Search for feedback loops was implemented with Code
segment S17, feed-forward loops with Code segment S18, bi-fan motifs with Code segment S19.
The results are summarized in Spreadsheet S28 and were visualized with ExcelTM (S4). The
number of islands does not consider highly connected nodes as single islands when they are not
included. For example, PKA, a highly connected node (k=49) is not included in the subnetworks
with lower connectivity threshold and is not considered an island by itself.
Computing the Density of Information Processing (DIP):
DIP is a measure of the local density of motifs and their interconnectedness within the interaction
space of the network. DIP is defined as:
M M i 1
GC i
DIPi = i
(1)
Li Li 1
Where M i = FBL3i + FBL 4 i + FFL3i + FFL 4 i + BIFAN i

Mi is the total number of feedback loops, feedforward loops and bifan motifs. Li is the total links and
i represent the step. FBL3 and FBL4 are feedback loops of size 3 and 4, FFL3 and FFL4 are
feedforward loops of size 3 and 4 and BIFAN are bi-fan motifs of size 4. GC is the grid coefficient
representing interconnectedness for the motifs, computed for the subnetwork at step i.
Computing the Motif Location Index (MLI):
MLI measure the concentration of specified motifs and various locations within the
network. MLI can vary from 0 to 1 depending on its relative distance from the extracellular ligands
to cellular machine, where 0 indicates location at the level of machines. MLI was calculated as
follows:
n

CPLM i

i =1 CPLM i + CPLLi
MLI =
(2)
n
where n is the size of the motif, CPLM is the characteristic path length from a node within the motif
to all other nodes in the cellular machine and CPLL is the characteristic path length from a node to
all extracellular ligands. If a node is an extracellular ligand then CPLL = 0; if the node is in the
plasma membrane CPLL= 1. If a node belongs to a cellular machine, CPLM = 0. The average
shortest path length was computed using Floyds algorithm (S5).
Supporting text
Functional Organization: From Components to Information Processing Motifs
Components within mammalian cells interact with one another to form local networks that
together form a single large network. This organization is essential for cellular components to
coordinate their individual activities and achieve the cohesiveness needed for cellular functions.
Information needs to flow between components in a continuous and organized manner. Determining
how this flow of information occurs is a crucial step in understanding the functional organization of
mammalian cells. This system of interacting cellular components based on phenotypic behavior
allows us to analyze the flow of information between the components to identify the emergence of
regulatory motifs that are capable of processing information as it flows through the network.
Regulatory motifs such as feedback loops and feedforward motifs allow the cell to process
information from extracellular signals, and decide when such information persists and when it is
transient. The presence of positive feedback loops arising from coupled biochemical reactions leads
to switching behavior that can enable state changes (S11). State change triggered by biochemical
switches has been demonstrated in developmental systems (S12).
The overall profile of motifs may be an initial indicator of the cells information processing
capability, when the quantitative and temporal characteristics of the interactions and consequent
motif formation are taken into account. Such quantitative specification may lead to comparative
profiles different from that observed here. Nevertheless, the identification and characterization of
motifs allows us to move from components and interactions to the next level of organization within
the cell.
Distinct regulatory motifs are active in response to signals from different ligands. The balance
of the emergent positive and negative motifs may define the capability of the ligand to induce
plasticity or maintain homeostasis. Comparison between motifs assumes that the motifs can be
formed and function concurrently. The relationship between the interaction steps in this
pseudodynamic analysis which considers propagation of functional connectivity over chemical
space and time steps needs to be specified at the level of individual interactions. This is discussed in

greater detail below. The relationship between distances in chemical space in such a functional
organization scheme and subcellular localization in terms of organelles and identified physical
compartments require further analysis to identify where the two types of organization overlap and
where they diverge.
At the functional level, each network configuration is likely to have its own distinct pattern of
regulatory motifs, and thus define capability of the cell for either maintaining homeostasis or
promoting state change such as that seen in synaptic plasticity. Would a large ensemble of such
states make the system intractably complex, such that we would not be able to either understand or
predict its behavior? This is unlikely because many of the states may have profiles of regulatory
motifs that produce convergent effects on cellular machines. Such convergence would provide a
molecular basis for redundancy of cellular function and endow the cellular system with robustness.
Functionally, cellular network states may fall into two broad categories: those that preserve
homeostasis during system perturbation and those that promote reorganization of the parts of the
cellular network. Such reorganization may include the synthesis of new components such as
transcription factors as well as other components that are required for expression of phenotypic
behavior. The notion that multiple network states may converge to yield a limited set of phenotypic
responses is supported by recent studies of networks of neurons where similar network behavior, as
defined by firing patterns, can arise from multiple network configurations based on individual
parameters that are quite different (S13). Perhaps such convergence represents a higher order of
organized redundancy arising from the many feedforward motifs. Thus, it is likely that cellular
systems are robust at varying levels of organization.
From Qualitative Representation to Quantitative Behavior of Motifs
Qualitative representations of cellular interaction such as those analyzed here do not take
into account individual reaction rates that define individual links. This simplifying assumption leads
to questions such as whether the identity and function of regulatory motifs would be preserved if the
reaction reactions for the links that make up the motifs vary over many orders of magnitude.
Experimental studies have shown that some of the links are relevant only at specific times, at
different perturbation, and at different locations within the cell. Such conditionality can be
manifested in many formats. Positive (also termed coherent) feedforward loops (FFL) can function
in several ways. For example, they can be coincidence detectors (AND gates), provide redundant
pathways to the same effectors (OR gates); they can lead to long sustained activity if one arm is fast
and the other arm is slower (S14, S15). These functions depend on the reaction rates of the two
arms. To obtain a preliminary idea of the potential validity of the motifs we have identified in this
study, we gathered a set of representative reactions from various classes (i.e., ligand-protein,
protein-protein, and enzymatic reactions) and evaluated their rates by comparing on and off rates
and Kcat values. The maximum observable difference was about 1000 fold (Table S2). We then
constructed a toy ODE model for motif 44 (Fig. S3). A schematic of the model is shown (Fig.
S11A). We varied the difference in rates between the two arms by a factor of up to 1000. Although
there are temporal differences in the output, the overall output profiles are quite similar within the
same time scale (Fig. S11B). These initial simulations suggest that the integrity of the motif is
persevered even with widely varying rates.
Although this toy ODE model has few components, it should be noted that this is likely to be
a limiting case since with multiple components in each arm, reactions rates are also likely to vary
and thus reducing the difference between the two arms. This is especially true for situations at twothree steps from the ligand, the location where the motifs become more prevalent. Thus, it is likely
that many of the motifs identified by qualitative analysis will be operative even when interaction
rates vary and the concentrations of components change in a regulated manner. Definitive proof that
6

each of these motifs is functional will only come when each reaction within a motif is parameterized
and input/output relationships are determined, and the existence of the motifs will be experimentally
verified.
Configuration of Motifs and choosing between homeostasis and plasticity:
The conversion of short term biochemical changes into long-term physiological changes including
state changes is often driven prolonged activation of key protein kinases and/or the activation or
induction of transcription factors. The extent and duration of activity of these downstream
components is regulated by the upstream signaling network. Since the network contains various
types of regulatory motifs, the balance between these motifs will determine the extent and duration
of the signal reaching these key components. Abundance of positive feed-forward or feedback loops
would evoke the extended activation of these components, while the presence of negative feedback
loops and gates could limit the duration of activation of the downstream components. Hence, the
ratio of the positive to negative feedback and feed-forward loops can determine whether or not
downstream components are activated for extended periods at sufficient levels. This extent and
duration of activation of the downstream components like the protein kinases or transcription factor
may in turn determine if the cell can maintain homeostatic behavior or undergo long-term
physiological changes. Thus, the ratio of positive to negative feedback and feed-forward motifs
could be a key indicator of whether, in the presence of external signals, the cell is configured to
maintain homeostatic balance or undergo state change. Since the propagation of connectivity by
different ligands involves different components and pathways, the ratio of the positive and negative
motifs is a function of the types of signals the cell receives and hence is conditional. Comparison of
the ratio of these motifs recruited per step for different ligands, glutamate, NE and BDNF clearly
demonstrates that the regulatory configuration is defined by the ligand that initiates the signal
propagation. Thus, by balancing the positive to negative motifs for different extracellular signals the
cell may be able to tune itself to either respond or not respond to extracellular signals by undergoing
long-term physiological changes. It is noteworthy that for both glutamate and BDNF in the
pathways to CREB the positive and negative loops are evenly balanced through nine steps. Such
balance of regulatory loops provides an explanation of how homeostasis within the cell can be
achieved even when a perturbing signal propagates through the system. The relative abundance of
the positive feedback and feedforward loops as compared to the negative loops in the sub-network
for NE to AMPA channels and to CREB is also noteworthy. These ratios may indicate that this subnetwork can hold and transfer information across time-scales leading to persistent changes. This
configuration of motifs might provide a systems level explanation of why the cAMP pathway is
associated with the late phase of long-term potentiation in the CA1 neuron (S16). It is also
noteworthy that propagation of connectivity from BDNF to AMPA channels and CREB results in
equal numbers of positive and negative feedback loops suggesting that BDNF would not play an
important role in changing the state of this cell. This is in agreement with experimental observation
that BDNF induces plasticity by affecting the functions of the presynaptic CA3 neuron (S17)
although acute effects of BDNF are observed in the postsynaptic CA1 neuron (S18). Thus some of
the characteristic responses evoked by important neurotransmitters may be understood by
identifying the configurations of the regulatory motifs they evoke within the cell.

Supporting figures
Fig. S1

Fig. S1 Visualization of the mammalian neuronal cellular network using the Pajek software
The network is visualized by placing nodes as triangles based in their functional compartments.
Size of triangles demonstrates the level of connectivity for the node. Green arrows represent
activation links, red arrows inhibition links, and blue arrows neutral links. The network data was
stored in the .net file format using custom code (Code segment S8). The output was then loaded into
the Pajek program (S2) for visualization.

Fig. S2
A

B
Statistical characteristics of the fully connected network:
Average links per node: 4.62
Characteristic Path Length: 4.21 (3.81 0.026)*
Grid Coefficient: 0.026 (0.005 0.0005)*
Clustering Coefficient: 0.11 (0.03 0.004)*
* Mean SD computed for 100 shuffled networks

Fig. S2 Statistics of the fully connected network

A) Distribution of interactions (links) into various categories. Interactions (links) in the cellular
network were sorted using two different criteria: the biochemical reaction and the effect of the link.
Upper pie chart shows sorting by biochemical reaction mechanism and the lower pie chart shows the
distribution of connections that activate or inhibit downstream components. Neutral links indicate
interactions where the directionality is unspecified. B) Overall network characteristics: In
parentheses are the mean and standard deviation of the characteristic path-lengths, clusteringcoefficients and grid-coefficients computed for 100 shuffled null networks with the same
connectivity distribution as the cellular network. These shuffled networks were created using an
approach based on Milo et al (S8). C) Log-log plot of the connectivity distribution. The number of
links per node (k) is plotted against the number of nodes with the same level of connectivity.

Fig. S3

Fig. S3 Schematic representation of the motifs identified within the cellular network
Network Motifs identified using the MFinder program. The figure visually represents the text based
output, produced by the MFinder program. These motifs are the most statistically significant
identified network motifs within the network. The MFinder program searches for motifs in directed
networks, it does not distinguish between positive and negative links. Thus, for this analysis we
considered positive and negative links as unidirectional and neutral links as bi-directional. The
MFinder program was developed by Alon and coworkers (S9). The network motifs of size 5 and 6
were identified using the sampling methods. The various types of motifs are identified by numbers.
The counts of the various types of motifs in the fully connected network are given in Table S1.

10

Table S1

Motifs counts
Motif #
CN*
SN**
Z-score
31
16
4.8 2.8
3.98
32
22
9.3 3.3
3.84
33
14
8.1 2.6
2.30
34
36
12.5 3.7
6.38
35
32
12.2 3.8
5.16
36
25
9.8 3.7
4.12
41
1011
186.6 32.6
25.31
42
108
68.2 14.8
2.69
43
26
7.2 4.8
3.91
44
303
104.0 15.5
12.88
45
57
17.8 7.3
5.39
46
105
40.0 10.9
5.97
47
49
31.5 8.3
2.12
* CN- Cellular Network.
** SN- Shuffled networks.
Mean SD computed for 100 shuffled networks.
Using the sampling method (S8):

Motif #
51
52
53
54
55
56
57
61
62
63
64
65
66
67

Motifs counts
CN*
SN**
0.254
0.023 0.025
0.065
0.002 0.003
0.662
0.005 0.011
0.048
0.000 0.002
0.100
0.003 0.007
0.020
0.0 0.0
0.016
0.0 0.0
0.395
0.001 0.011
0.189
0.0 0.0
0.050
0.0 0.0
0.096
0.0 0.0
0.070
0.0 0.0
0.059
0.0 0.0
0.049
0.0 0.0

Z-score
9.27
11.42
60.05
26.57
13.92
NA
NA
36.01
NA
NA
NA
NA
NA
NA

Table S1 Counts of the various types of motifs found in the cellular network
Motifs were counted using the MFinder program (S9). Counts for the motifs in the cellular network
(CN) are compared to the mean for 100 control (shuffled) networks. Z-score indicates the statistical
significance for the number found in the real network as compared to the mean of the control
networks. Motifs of size 5 and 6 were estimated using the sampling method described by Kashtan et
al. (S9). Estimated values are given as fractions (count of the specified motifs / the total number of
motifs of the same size).

11

Fig. S4
A

Fig. S4 Analysis of subnetworks from glutamate, NE and BDNF

The number of links (A) and nodes (B) added per step, downstream from three ligands known to
regulate plasticity in hippocampal neurons. C) Characteristic Path Length (CPL), D) Clustering
Coefficient (CC) and E) Grid Coefficient (GC) computed for subnetworks emanating from
glutamate, nor-epinephrine and BDNF.
Fig. S5
A

Fig. S5 Comparison of positive vs. negative feed-forward loop motifs in subnetworks

emanating in steps from the three ligands: glutamate, nor-epinephrine and BDNF
Counts were obtained using the source code described under Methods. Positive feed-forward loop
motifs are feed-forward loops where both arms to the target node are positive. If at least one arm
is negative the feed-forward is considered negative. Positive arm is defined as an arm with
either no negative links or even number of negative links.

12

Fig. S6

Fig. S6 Regulatory motifs identified in subnetworks emanating in steps from glutamate (Glu),
norepinephrine (NE) and BDNF
The motifs identified using our algorithms (see custom codes S17-S19) can classify motifs based on
the different types of links and nodes. This is an enhancement to the MFinder program that only
identifies more abstract motifs based on directionality only.
A) The scaffold motif is made of three nodes connected solely by neutral links.
A-I: A schematic illustration of a scaffold motif.
A-II: The counts of scaffold motifs found in subnetworks generated from the three ligands
A-III: Representative examples of the scaffold motifs.
B) The bi-fan motif
B-I: Schematic representation of the bifan motif.
B-II: The counts of bi-fan motifs found in subnetworks generated from the three ligands.
B-III: Representative examples of bifan motifs.
C) The feedforward loop motifs are composed of three or four nodes connected with directed links
only where a source node feeds into a target node through two alternative routes.
C-I: Schematic representations of the feedforward loops

13

C-II: The counts of feed-forward loop motifs found in the subnetworks generated from the three
ligands
C-III: Representative examples of feed-forward motifs.
Positive feed-forward loop motifs are feed-forward loops where both arms to the target node are
positive. Positive arm is defined as an arm with either no negative links or even number of
negative links. If at least one arm is negative the feed-forward is considered negative.
Abbreviations: PKA: protein kinase A, PKC: protein kinase C, CaM: calmodulin, CaN: calcineurin,
GlyR: glycine receptor, NMDAR: NMDA receptor, AC2: adenylyl cyclase 2, PLC: phospholipase
C beta, CREB: cyclic-AMP response element binding protein. CREM: cyclic-AMP response
element modulator. -ARK: beta adrenergic receptor kinase.
Fig. S7
A

Fig. S7 Size of subnetworks from extracellular ligands to specified effectors

A) Schematic representation of the source and target nodes for the subnetworks starting from the
three extracellular ligands: glutamate, nor-epinephrine (NE) and brain-drived neurotrophic factor
(BDNF), (source nodes) to reach the effectors AMPA receptor-channels (AMPAR) or the
transcription factor cyclic-AMP response element binding protein (CREB) (target nodes). B) The
changes in the number of nodes as more steps are allowed to reach the target nodes (effectors) from
the source ligands for six sets of subnetworks.

14

Fig S8
A

Fig. S8 Feedback and feed-forward loops in subnetworks from extracellular ligands to CREB
A) Schematic of the source and target nodes for the subnetworks starting from the three extracellular
ligands: glutamate, nor-epinephrine (NE) and brain-derived neurotrophic factor (BDNF), (source
nodes) to reach the effector CREB (target node). B-C) Counts of the number of total three and four
component positive and negative feedback and feedforward loops, in subnetworks where more steps
are gradually allowed between the source and target nodes.

15

Fig. S9
A

Fig. S9 Subnetworks characteristics from extracellular ligands to AMPAR

A) Schematic of the source and target nodes for the subnetworks starting from the three extracellular
ligands: glutamate, nor-epinephrine (NE) and brain-derived neurotrophic factor (BDNF), (source
nodes) to reach the effector AMPA channel/receptor (AMPAR) (target node). B-D) Changes in the
number of links as more steps are allowed to reach the effectors from each ligand are compared to
the same analysis with shuffled networks. Only the directionality of the links that do not involve the
ligands or the effectors was randomly swapped while preserving the connectivity. The resultant
graphs for both the cellular network and shuffled networks were curve fitted. For all cellular
subnetworks the best fit function was linear (R2 =0.99-0.96). Individual values are given as lin R2.
For all of the shuffled networks the best fit was obtained for either exponential or power law
functions (lin and pow respectively). E-G) Counts of the number of total three and four component
positive and negative feedback and feedforward loops, in subnetworks where more steps are
gradually allowed between the source and target nodes.

16

Fig. S10

Fig. S10 Changes in network size and statistical properties with gradual incorporation of
nodes based on nodal connectivity.
Sub-networks were created including only nodes with specified number of links per node (X-axis).
A) The number of islands (isolated clusters of nodes that are not connected to one another), nodes,
and links were counted in each subnetwork. B) Characteristic path-length (CPL) and clustering
coefficients (CC), grid coefficients (GC) were calculated for each subnetwork.

17

Fig. S11
A
input

Activated A

Activated B

output
Negative
Regulator

B
kcat
kcat
kcat
kcat

0.10

A/kcat
A/kcat
A/kcat
A/kcat

B=
B=
B=
B=

1/1
10/1
10/0.1
10/0.01

0.15

Output

Output

0.15

0.05

Km
Km
Km
Km

0.10

A/Km
A/Km
A/Km
A/Km

B=
B=
B=
B=

1/1
10/1
10/0.1
10/0.01

0.05

0.00

0.00

500

1000

1500

500

1000

1500

Fig. S11 Quantitative evaluation of a simple feedforward motif (motif 44 in Fig. S3).
A) A feedforward motif (Motif 44, Fig S3) was constructed and simulated using a system of coupled
ODEs. In this model, a simulated square pulse of input activates enzymes A and B. After 300
seconds, the input was washed out, and the behavior of the output, which is activated by both A
and B, was studied. To balance the activities of the activator, a negative regulator was included.
The initial concentration of all components was 1 M. B) To study the motif behavior, sensitivity
analyses were performed on kcat and Km. The ratio between the kinetics parameters of the two arms
were varied from 10 (kcatA = 10 s-1, kcatB = 1 s-1), to 1000 (kcatA = 10 s-1, kcatB = 0.01 s-1). Similar
analysis was performed on Km. The activity of the output component is plotted as a function of
time. The analysis shows that the motif behavior is robust. Changing the kinetics parameters over 3
orders of magnitude had little effect on the qualitative behavior of the motif. As assessed by inputoutput relationships. These initial results suggests that components with widely varying reactions
rates can come together to form functional units.

18

Table S2
Molec#1/Catalyst

Molec#2/Substrate/Product

Reaction type

kf
(/M/s)

kr (/s)

KD/KM
(M)

kcat (/s)

Ref.

Rap1GAPII

Rap1

GAP

1.4

80.5

65

11

(1)

cAMP

PKA site1

Binding

1.67

0.0167

0.010

(2)

cAMP

PKA site2

Binding

0.0093

0.00028

0.030

(2)

ARK

-AR

Phosphorylation

Csk

Src

Phosphorylation

1.8

40

0.2

0.2

(3)

22

0.4

(4)

calcium

N-term calmodulin

Binding

500

(5)

calcium

C-term calmodulin

High Aff-Binding

10

(5)

calcium

C-term calmodulin

Low Aff-Binding

100

(5)

MKP3

ERK2

Dephosphorylation

CDK1-cyclin Clb2

unidentified substrate/s

PIP2

PKB (PH dom)

Binding

0.022

0.2

(6)

35
(ATP)

2.2

(7)

0.5

RasGAP

Ras

GAP

4.8

9.12

(9)

Caspase3

Cleavage

0.089

0.0168

(10)

Caspase 3

PARP

Cleavage

5.6

0.213

(10)

Sos

Ras

GEF

0.50505

0.02

(11)

PKA

Representative substrate

Phosphorylation

2.4

2.7

(12)

PKA

Representative substrate

Phosphorylation

7.5

(13)

Tau

Phosphorylation

33

0.0433

(14)

Tau

Phosphorylation

27

0.217

(14)

Dephosphorylation

3.8

0.41

(15)

Dephosphorylation

1.6

0.2

(15)

Calcineurin
Calcineurin

G-substrate, a substrate for

PKG
DARPP-32, a substrate for
PKA

Calcineurin

synapsin I (site 1)

Dephosphorylation

0.053

(15)

Calcineurin

synapsin I (site 2)

Dephosphorylation

4.4

0.04

(15)

Grb2

Sos

Binding

0.025

0.0168

PKC (Ca bound)

DAG

Binding

0.008

8.6348

(16)

calmodulin

MLCK (non phosphorylated)

Binding

28

0.031

(17)

calmodulin

MLCK (non phosphorylated)

Binding

0.186

(17)

MEK

MAPK

Phosphorylation

0.476

0.113

(18,
19)

Raf

MEK1

Phosphorylation

0.8

0.105

(20)

GSK3

-CATENIN

Phosphorylation

0.1276

3.5

(21,
22)

1.7

6.13

(23)

Used crude cell lysate as

substrate. i.e. no single
target, but perhaps more
physiological

(16)

STEP

MAPK

Dephosphorylation

38

12

AC

cAMP

Synthesis

1.4

7.9

(24,
25)
(26,
27)

PDE4D

cAMP

Cleavage

NWASP

Arp2/3

Binding

0.003

(28)

Grb2

NWASP

Binding

0.005

(28)

19

2-fold adjustment for T;

KD & kr are lower limits
high and low-affinity
binding @ C-term reflects
CaM complexing;
dissociation but not
association rate constants
were modified by CaM's
interactions

(8)

Caspase 9

CDK5 (p35
isoform)
CDK5 (p25
isoform)

Notes

Km = 0.476 uM for 1st

phosphorylation, 0.046
uM for second
phosphorylation

AC 1

References for Table S2:

1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.

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Kim, C. M., Dion, S. B. & Benovic, J. L. (1993) J Biol Chem 268, 15412-8.
Lieser, S. A., Shindler, C., Aubol, B. E., Lee, S., Sun, G. & Adams, J. A. (2005) J Biol Chem 280, 7769-76.
Gaertner, T. R., Putkey, J. A. & Waxham, M. N. (2004) J Biol Chem 279, 39374-82.
Zhou, B., Wang, Z. X., Zhao, Y., Brautigan, D. L. & Zhang, Z. Y. (2002) J Biol Chem 277, 31818-25.
Ubersax, J. A., Woodbury, E. L., Quang, P. N., Paraz, M., Blethrow, J. D., Shah, K., Shokat, K. M. & Morgan, D. O. (2003)
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Table S2 Reaction rates for representative reactions underlying interactions in the network
and a listing of the primary references from which these rates were obtained
Rates for several classes of the reactions underlying the links in the network were collected from
published studies. The reactions for which rates are shown include: non-covalent binding including
ligand-protein and protein-protein interactions, enzymatic reactions such as cAMP synthesis,
phosphorylation, dephosphorylation, proteolytic cleavage, and changes in catalytic activity resulting
from protein-protein interactions such as the GEF and GAP activity for GTPases. These types of
reactions account for a large portion of the interactions within the cellular network.

20

Table S3
File name
Text file S1
Text file S2
Text file S3
Text file S4
Text file S5
Text file S6
Code segment S7
Code segment S8
Code segment S9
Code segment S10
Code segment S11
Code segment S12
Code segment S13
Code segment S14
Code segment S15
Code segment S16
Code segment S17
Code segment S18
Code segment S19
Movie S20
Movie S21
Movie S22
Spreadsheet S23
Spreadsheet S24
Spreadsheet S25
Spreadsheet S26
Spreadsheet S27
Spreadsheet S28

Description
Network data in flat file format.
Pajek format text file describing the network data and assigns 2D location for nodes on the map.
Raw output from the MFinder program searching for motifs of size 3.
Raw output from the MFinder program searching for motifs of size 4.
Raw output from the MFinder program searching for motifs of size 5.
Raw output from the MFinder program searching for motifs of size 6.
Rules for sorting components into functional machines.
Converts the network into a Pajek format text file.
Creates sub-network where connectivity is propagated from ligands.
Analysis for nodes, interactions, motifs, clustering and path-lengths of sub-networks starting at
ligands.
Implementation of the Floyd algorithm to compute the characteristic path-length.
Implementation of the clustering coefficient algorithm.
Implementation of the grid coefficient algorithm.
Algorithm used to create 100 shuffled networks with the same connectivity distribution and one
island.
Code segment used to create sub-network from specified source node to specified target node.
Code used to create sub-networks with gradual allowable nodal connectivity.
Algorithm used to search for feedback loops.
Algorithm used to search for feed-forward loops.
Algorithm used to search for bi-fan motifs.
Illustration of the breadth-first concept.
Illustration of the sub-networks where the source and target are specified.
Illustration of the concept of gradual inclusion of nodes based on connectivity.
Connectivity propagation per step from all ligands.
Number of nodes, interactions, motifs, computed clustering and path-lengths for sub-networks
starting at ligands.
Connectivity distribution table.
Characteristic path-lengths, clustering coefficients and grid coefficients for the real network and
for 100 shuffled networks with the same connectivity distribution.
Results of the analysis of sub-networks from source to target.
Analysis of sub-networks based on nodal connectivity.

Table 3 Supporting external files

The network database is provided in Text file S1 as a flat file where the field separator is a blank
space. Each line in the file describes an interaction extracted from a specific journal. The tokens
description is provided in the methods section under Storage of the network data. The Pajek
software can be used to visualize the network with Text file S2 (S2). Text files S3-S6 are the raw
output files from the MFinder program. Code segments S7-S19 contain algorithms written in C/C++
used to generate, analyze and visualize the subnetworks in the study. Movies S20-S23 illustrate the
concept we termed pseudodynamics used to create the three different types of subnetworks.
Spreadsheets S23-S28 contain the raw data from the analysis. These tables were used to create most
of the graphs in the study. The supporting external files can be found also at
http://amp.pharm.mssm.edu/network/sm/SM.htm and at http://www.mssm.edu/labs/iyengar.
Other supporting online materials are available at: http://amp.pharm.mssm.edu/network/index.htm

21

Supporting References:
S1. N. R. Gough, L. B. Ray, Science STKE. 135, EG8 (2002)
S2. http://vlado.fmf.uni-lj.si/pub/networks/pajek/ (2004)
S3. http://www.mathworks.com/products/matlab/ (2004)
S4. http://office.microsoft.com/excel/
(2004)
S5. T. H. Cormen, et al. 2002, Introduction to Algorithms, MIT Press Cambridge, MA.
S6. D. J. Watts, S. H. Strogatz, Nature. 393, 440 (1998)
S7. G. R. Caldarelli, Pastor-Satorras et al. European Physical Journal B. 38, 183 (2004)
S8. R. Milo, S. Shen-Orr, S. Itzkovitz, et al., Science. 298, 824 (2002)
S9. N. Kashtan, S. Itzkovitz, R. Milo, Bioinformatics. 20, 1746 (2004)
S10. http://office.microsoft.com/visio (2004)
S11. U.S. Bhalla, R. Iyengar, Science. 283, 381 (1999)
S12. W. Xiong , J. E, Ferrell Jr. Nature. 426, 460 (2003)
S13. A.A. Prinz AA, D. Bucher, E. Marder, Nature Neuroscience 7, 1345 (2004)
S14. S. Mangan, U. Alon, Proc Natl Acad Sci U S A. 100, 11980 (2003)
S15. S. Mangan, A. Zaslaver, U. Alon, J Mol Biol. 334, 197 (2003)
S16 P. V. Nguyen, T. Abel, E. R. Kandel, Science. 265, 1104 (1994).
S17. S. S. Zakharenko, S. L. Patterson, I. Dragatsis, et al., Neuron. 39, 975 (2003).
S18. Y. Kovalchuk, E. Hanse, K. W. Kafitz, et al., Science. 295, 1729 (2002).
Author Contributions
AM assembled the large-scale network, wrote all of the custom-code and conducted all of
the analysis described in this study. SLJ developed the database to manage and maintain the
components links and references programs; AM, SN, AH, EG, BD-T, NJE, SLJ, RDB and RI found
and read the original papers cited as references to verify the components and interactions. GW and
PTR developed the original connections map of nearly 200 components on which the network used
in this study was based upon. JJR, AK and GAS provided approaches to analyze and statistically
validate several of the key findings. NJE performed the quantitative analysis of the feedforward
motif. AH assembled the table of rate constants. RDB provided supervision of the development of
interactions maps and its anchoring to the hippocampal neuron. RI was responsible for overall
supervision of the project including analysis of the data and writing of the manuscript and is
responsible for the final version of the submitted paper.

22