Biochemical Compartmentalization Research Articles

Spiny and Non-spiny Parvalbumin-Positive Hippocampal Interneurons Show Different Plastic Properties

SummaryDendritic spines control synaptic transmission and plasticity by augmenting post-synaptic potentials and providing biochemical compartmentalization. In principal cells, spines cover the dendritic tree at high densities, receive the overwhelming majority of excitatory inputs, and undergo experience-dependent structural re-organization. Although GABAergic interneurons have long been considered to be devoid of spines, a number of studies have reported the sparse existence of spines in interneurons. However, little is known about their organization or function at the cellular and network level. Here, we show that a subset of hippocampal parvalbumin-positive interneurons forms numerous dendritic spines with highly variable densities and input-selective organization. These spines form in areas with reduced perineuronal net sheathing, predispose for plastic changes in protein expression, and show input-specific re-organization after behavioral experience.

Controllable Protein Phase Separation and Modular Recruitment to Investigate Biochemical Compartmentalization in Membraneless Organelles

Many intrinsically disordered proteins (IDPs) self-assemble via liquid-liquid phase separation into spherical droplets, which function as membraneless organelles with critical cellular functions. Despite significant interest, fundamental questions remain unanswered about the functional capabilities of membraneless organelles. It is well established that membrane-bound organelles provide aqueous compartments adapted for specific biochemical reactions, but it is unclear what roles membraneless organelles play in regulating the kinetics of intracellular reactions. The field has been hindered because few tools exist to systematically investigate the consequences of localizing enzymes and substrates to membraneless organelles. Therefore, we manipulated the intrinsically disordered, arginine/glycine-rich RGG domain from the P granule protein LAF-1 to demonstrate controllable phase separation and cargo recruitment to a synthetic membraneless compartment. First, we demonstrated methods to control phase behavior by externally triggering droplet assembly and disassembly. This was accomplished by using specific proteases to manipulate the valency of IDP domains and presence of solubility-enhancing domains. Second, we characterized permeability of these compartments to soluble macromolecules and devised strategies to target and colocalize cargo molecules into the droplets. Soluble cargos were recruited using either RGG domains or coiled-coiled interaction domains as recruitment modules, and cargo release was triggered by proteolytic removal of the recruitment domains. Droplet assembly and cargo recruitment were robust and occurred in cytoplasm. Our results using this platform suggest it is now possible to controllably recruit multiple enzymes and substrates to stimulus-responsive membraneless organelles. This system provides a much-needed experimental framework to answer unsolved problems in the biophysics of membraneless organelles and to harness IDP compartments for bioengineering applications.

Desmosomal cadherin association with a dynein‐cortactin complex promotes actin rearrangements required for epidermal morphogenesis

The epidermis is a multi‐layered, renewing epithelium that serves as an essential barrier against water loss and environmental insults in vertebrates. Its morphogenesis occurs through a tightly regulated program of biochemical and architectural changes in which epidermal keratinocytes in the basal layer commit to differentiate, and move towards the skin's surface in a process called stratification. During this process, tissue integrity is ensured by intercellular junctions called desmosomes, which anchor the intermediate filament cytoskeleton to sites of desmosomal cadherin‐mediated adhesion. In contrast to the adherens junction protein, E‐cadherin, which is expressed in all epidermal layers, seven desmosomal cadherins are expressed in a highly patterned differentiation‐dependent manner in epidermis. This genomic complexity and spatial patterning of desmosome components in stratified tissues suggests that they do more than perform textbook functions in mediating cell‐cell adhesion. Here, we reveal an unexpected role for the cadherin desmoglein 1 (Dsg1) in remodeling the actin cytoskeleton to promote the transit of basal cells into suprabasal layers through a process of delamination/extrusion. This function requires a newly recognized association between Dsg1 and the dynein light chain, Tctex‐1, and actin scaffolding protein, cortactin. We demonstrate that Tctex‐1, as a part of the dynein complex, controls the proper biochemical compartmentalization and segregation of Dsg1‐containing desmosomes from adherens junctions. Moreover, uncoupling the Dsg1‐Tctex‐1 complex from dynein or abrogation of Dsg1‐Tctex‐1 interactions interfered with the proper positioning of cortactin, which in turn is required to recruit the Arp2/3 complex and promote F‐actin network reorganization. Assembly of this molecular complex at the onset of Dsg1 expression during differentiation in cells already containing E‐cadherin results in decreased tension at adherens junctions, as measured through an E‐cadherin FRET sensor and accumulation of the mechanosensitive molecule vinculin. The proper coordination of this chain of events is required for the efficient delamination of basal cells in an organotypic model of tissue development. Thus, Dsg1 serves as a scaffold that exerts spatial control over mechanical changes necessary to promote early stages of epidermal morphogenesis.Support or Funding InformationSupported by NIH R01AR041836, R37AR043380, R01CA122151, the Dermatology Foundation and J.L. Mayberry Endowment.

Nuclear Export as a Novel Therapeutic Target: The CRM1 Connection.

The integrity of eukaryotic cellular function depends on molecular and biochemical compartmentalization. The transport of macromolecules between compartments requires specific and energydriven mechanisms. It occurs through a class of transport proteins known as karyopherins, which are divided in three different groups (exportins, importins, and transportins). The ubiquitous exportin Chromosome Region Maintenance 1 (CRM1) is involved in the transport of many proteins and RNA molecules from nucleus to cytoplasm. We have reviewed the available evidence supporting the relevance of CRM1 in the biology of several human neoplasms, its potential role in drug resistance, and its promise as a therapeutic target. Here we discuss different cancer related proteins (tumor suppressor genes, oncogenes, and enzymatic therapeutic targets), their function, and their association with CRM1, as well as agents that specifically inhibit CRM1, their mechanism of action, and their clinical relevance in certain human neoplasms. The directionality of nuclear transport and the specific molecular cargo in question are of paramount importance when examining the effects that CRM1 inhibition may have on cellular pathophysiology. The available data point out the potential role of CRM1-dependent nuclear export of regulatory proteins in the biology of certain human malignancies. Further studies should expand and clarify the importance of this mechanism in the pathobiology of human neoplasia.

Substance P Weights Striatal Dopamine Transmission Differently within the Striosome-Matrix Axis.

The mammalian striatum has a topographical organization of input-output connectivity, but a complex internal, nonlaminar neuronal architecture comprising projection neurons of two types interspersed among multiple interneuron types and potential local neuromodulators. From this cellular melange arises a biochemical compartmentalization of areas termed striosomes and extrastriosomal matrix. The functions of these compartments are poorly understood but might confer distinct features to striatal signal processing and be discretely governed. Dopamine transmission occurs throughout striosomes and matrix, and is reported to be modulated by the striosomally enriched neuromodulator substance P. However, reported effects are conflicting, ranging from facilitation to inhibition. We addressed whether dopamine transmission is modulated differently in striosome-matrix compartments by substance P.We paired detection of evoked dopamine release at carbon-fiber microelectrodes in mouse striatal slices with subsequent identification of the location of recording sites with respect to μ-opioid receptor-rich striosomes. Substance P had bidirectional effects on dopamine release that varied between recording sites and were prevented by inhibition of neurokinin-1 receptors. The direction of modulation was determined by location within the striosomal-matrix axis: dopamine release was boosted in striosome centers, diminished in striosomal-matrix border regions, and unaffected in the matrix. In turn, this different weighting of dopamine transmission by substance P modified the apparent center-surround contrast of striosomal dopamine signals. These data reveal that dopamine transmission can be differentially modulated within the striosomal-matrix axis, and furthermore, indicate a functionally distinct zone at the striosome-matrix interface, which may have key impacts on striatal integration.

Structural and functional studies of membrane remodeling machines.

Building cells from their component parts will hinge upon our ability to reconstitute biochemical compartmentalization and exchange between membrane-delimited organelles. By contrast with our understanding of other cellular events, the mechanisms that govern membrane trafficking has lagged because the presence of phospholipid bilayers complicates the use of standard methods. This chapter describes in vitro methods for purifying, reconstituting, and visualizing membrane remodeling activities directly by electron cryomicroscopy.

Spine neck plasticity regulates compartmentalization of synapses

Dendritic spines have been proposed to transform synaptic signals through chemical and electrical compartmentalization. However, the quantitative contribution of spine morphology to synapse compartmentalization and its dynamic regulation are still poorly understood. We used time-lapse super-resolution stimulated emission depletion (STED) imaging in combination with fluorescence recovery after photobleaching (FRAP) measurements, two-photon glutamate uncaging, electrophysiology and simulations to investigate the dynamic link between nanoscale anatomy and compartmentalization in live spines of CA1 neurons in mouse brain slices. We report a diversity of spine morphologies that argues against common categorization schemes and establish a close link between compartmentalization and spine morphology, wherein spine neck width is the most critical morphological parameter. We demonstrate that spine necks are plastic structures that become wider and shorter after long-term potentiation. These morphological changes are predicted to lead to a substantial drop in spine head excitatory postsynaptic potential (EPSP) while preserving overall biochemical compartmentalization.

Synaptic amplification by dendritic spines enhances input cooperativity

Dendritic spines are the nearly ubiquitous site of excitatory synaptic input onto neurons1–2 and as such are critically positioned to influence diverse aspects of neuronal signaling. Decades of theoretical studies have proposed that spines may function as highly effective and modifiable chemical and electrical compartments that regulate synaptic efficacy, integration, and plasticity3–8. Experimental studies have confirmed activity-dependent structural dynamics and biochemical compartmentalization by spines9–12. However, a longstanding debate remains over the influence of spines on the electrical aspects of synaptic transmission and dendritic operation3–8,13–18. Here, we measured the amplitude ratio (AR) of spine head to parent dendrite voltage across a range of dendritic compartments and calculated the associated Rneck for spines at apical trunk dendrites in hippocampal CA1 pyramidal neurons. We found that Rneck is large enough (~500 MΩ) to substantially amplify the spine head depolarization associated with a unitary synaptic input by ~1.5- to ~45-fold depending on parent dendritic impedance. A morphologically realistic compartmental model capable of reproducing the observed spatial profile of AR indicates that spines provide a consistently high impedance input structure throughout the dendritic arbor. Finally, we demonstrate that the amplification produced by spines encourages electrical interaction among coactive inputs through an Rneck-dependent increase in spine head voltage- dependent conductance activation. We conclude that the electrical properties of spines promote nonlinear dendritic processing and associated forms of plasticity and storage, thus fundamentally enhancing the computational capabilities of neurons19–21.

Cav2.1 in Cerebellar Purkinje Cells Regulates Competitive Excitatory Synaptic Wiring, Cell Survival, and Cerebellar Biochemical Compartmentalization

In the adult cerebellum, each Purkinje cell (PC) is innervated by a single climbing fiber (CF) in proximal dendrites and 10(5)-10(6) parallel fibers (PFs) in distal dendrites. This organized wiring is established postnatally through heterosynaptic competition between PFs and CFs and homosynaptic competition among multiple CFs. Using PC-specific Cav2.1 knock-out mice (PC-Cav2.1 KO mice), we have demonstrated recently that postsynaptic Cav2.1 plays a key role in the homosynaptic competition by promoting functional strengthening and dendritic translocation of single "winner" CFs. Here, we report that Cav2.1 in PCs, but not in granule cells, is also essential for the heterosynaptic competition. In PC-Cav2.1 KO mice, the extent of CF territory was limited to the soma and basal dendrites, whereas PF territory was expanded reciprocally. Consequently, the proximal somatodendritic domain of PCs displayed hyperspiny transformation and fell into chaotic innervation by multiple CFs and numerous PFs. PC-Cav2.1 KO mice also displayed patterned degeneration of PCs, which occurred preferentially in aldolase C/zebrin II-negative cerebellar compartments. Furthermore, the mutually complementary expression of phospholipase Cβ3 (PLCβ3) and PLCβ4 was altered such that their normally sharp boundary was blurred in the PCs of PC-Cav2.1 KO mice. This blurring was caused by an impaired posttranscriptional downregulation of PLCβ3 in PLCβ4-dominant PCs during the early postnatal period. A similar alteration was noted in the banded expression of the glutamate transporter EAAT4 in PC-Cav2.1 KO mice. Therefore, Cav2.1 in PCs is essential for competitive synaptic wiring, cell survival, and the establishment of precise boundaries and reciprocity of biochemical compartments in PCs.

Magnetic Resonance Microscopy Contribution to Interpret High‐Resolution Magic Angle Spinning Metabolomic Data of Human Tumor Tissue

HRMAS NMR is considered a valuable technique to obtain detailed metabolic profile of unprocessed tissues. To properly interpret the HRMAS metabolomic results, detailed information of the actual state of the sample inside the rotor is needed. MRM (Magnetic Resonance Microscopy) was applied for obtaining structural and spatially localized metabolic information of the samples inside the HRMAS rotors. The tissue was observed stuck to the rotor wall under the effect of HRMAS spinning. MRM spectroscopy showed a transference of metabolites from the tissue to the medium. The sample shape and the metabolite transfer after HRMAS indicated that tissue had undergone alterations and it can not be strictly considered as intact. This must be considered when HRMAS is used for metabolic tissue characterization, and it is expected to be highly dependent on the manipulation of the sample. The localized spectroscopic information of MRM reveals the biochemical compartmentalization on tissue samples hidden in the HRMAS spectrum.

Expression of the small heat shock protein family in the mouse CNS: Differential anatomical and biochemical compartmentalization

The small heat shock proteins (sHsps) are a family of molecular chaperones defined by an alpha-crystallin domain that is important for sHsps oligomerization and chaperone activity. sHsps perform many physiological functions including the maintenance of the cellular cytoskeleton, the regulation of protein aggregation and modulate cell survival in a number of cell types including glial and neuronal cells. Many of these functions have been implicated in disease processes in the CNS and indeed sHsps are considered targets for disease therapy. Despite this, there is no study that systematically and comparatively characterized sHsps expression in the CNS. In the present study we have analyzed the expression of this gene family in the mouse brain by reverse-transcriptase polymerase chain reaction (RT-PCR), in situ hybridization and Western blotting. Gene expression analysis of the 10 known members of mammalian sHsps confirms the presence of 5 sHsps in the CNS. A distinct white matter specific expression pattern for HspB5 and overlapping expression of HspB1 and HspB8 in the lateral and dorsal ventricles of the brain is observed. We confirm protein expression of HspB1, HspB5, HspB6 and HspB8 in the brain. Further subcellular fractionation of brain and synaptosomes details a distinct subcompartment-specific association and detergent solubility of sHsps. This biochemical signature is indicative of an association with synaptic and other neural specializations. This observation will help one understand the functional role played by sHsps during physiology and pathology in the CNS.

Dendritic spines linearize the summation of excitatory potentials

In mammalian cortex, most excitatory inputs occur on dendritic spines, avoiding dendritic shafts. Although spines biochemically isolate inputs, nonspiny neurons can also implement biochemical compartmentalization; so, it is possible that spines have an additional function. We have recently shown that the spine neck can filter membrane potentials going into and out of the spine. To investigate the potential function of this electrical filtering, we used two-photon uncaging of glutamate and compared the integration of electrical signals in spines vs. dendritic shafts from basal dendrites of mouse layer 5 pyramidal neurons. Uncaging potentials onto spines summed linearly, whereas potentials on dendritic shafts reduced each other's effect. Linear integration of spines was maintained regardless of the amplitude of the response, distance between spines (as close as < 2 microm), distance of the spines to the soma, dendritic diameter, or spine neck length. Our findings indicate that spines serve as electrical isolators to prevent input interaction, and thus generate a linear arithmetic of excitatory inputs. Linear integration could be an essential feature of cortical and other spine-laden circuits.

The spine neck filters membrane potentials

Dendritic spines receive most synaptic inputs in the forebrain. Their morphology, with a spine head isolated from the dendrite by a slender neck, indicates a potential role in isolating inputs. Indeed, biochemical compartmentalization occurs at spine heads because of the diffusional bottleneck created by the spine neck. Here we investigate whether the spine neck also isolates inputs electrically. Using two-photon uncaging of glutamate on spine heads from mouse layer-5 neocortical pyramidal cells, we find that the amplitude of uncaging potentials at the soma is inversely proportional to neck length. This effect is strong and independent of the position of the spine in the dendritic tree and size of the spine head. Moreover, spines with long necks are electrically silent at the soma, although their heads are activated by the uncaging event, as determined with calcium imaging. Finally, second harmonic measurements of membrane potential reveal an attenuation of somatic voltages into the spine head, an attenuation directly proportional to neck length. We conclude that the spine neck plays an electrical role in the transmission of membrane potentials, isolating synapses electrically.

Repeated Stress Induces Dendritic Spine Loss in the Rat Medial Prefrontal Cortex

The prefrontal cortex (PFC) plays an important role in higher cognitive processes, and in the regulation of stress-induced hypothalamic-pituitary-adrenal (HPA) activity. Here we examined the effect of repeated restraint stress on dendritic spine number in the medial PFC. Rats were perfused after receiving 21 days of daily restraint stress, and intracellular iontophoretic injections of Lucifer Yellow were carried out in layer II/III pyramidal neurons in the anterior cingulate and prelimbic cortices. We found that stress results in a significant (16%) decrease in apical dendritic spine density in medial PFC pyramidal neurons, and confirmed a previous observation that total apical dendritic length is reduced by 20% in the same neurons. We estimate that nearly one-third of all axospinous synapses on apical dendrites of pyramidal neurons in medial PFC are lost following repeated stress. A decrease in medial PFC dendritic spines may not only be indicative of a decrease in the total population of axospinous synapses, but may impair these neurons' capacity for biochemical compartmentalization and plasticity in which dendritic spines play a major role. Dendritic atrophy and spine loss may be important cellular features of stress-related psychiatric disorders where the PFC is functionally impaired.

Quantitative Analysis of Microtubule Transport in Growing Nerve Processes

In neurons, tubulin is synthesized primarily in the cell body [1], whereas the molecular machinery for neurite extension and elaboration of microtubule (MT) array is localized to the growth cone region. This unique functional and biochemical compartmentalization of neuronal cells requires transport mechanisms for the delivery of newly synthesized tubulin and other cytoplasmic components from the cell body to the growing axon. According to the polymer transport model, tubulin is transported along the axon as a polymer. Because the majority of axonal MTs are stationary at any given moment [2], it has been assumed that only a small fraction of MTs translocates along the axon by saltatory movement reminiscent of the fast axonal transport. Such intermittent “stop and go” MT transport has been difficult to detect or to exclude by using direct video microscopy methods. In this study, we measured the translocation of MT plus ends in the axonal shaft by expressing GFP-EB1 in Xenopus embryo neurons in culture. Formal quantitative analysis of MT assembly/disassembly indicated that none of the MTs in the axonal shaft were rapidly transported. Our results suggest that transport of axonal MTs is not required for delivery of newly synthesized tubulin to the growing nerve processes.

On the electrical function of dendritic spines

Dendritic spines mediate most excitatory inputs in the brain, yet their function is still unclear. Imaging experiments have demonstrated their role in biochemical compartmentalization at individual synapses, yet theoretical studies have suggested that they could serve an electrical function in transforming synaptic inputs and transmitting dendritic spikes. Recent data indicate that spines possess voltage-dependent conductances and that these channels can be spine-specific. Although direct experimental investigations of the electrical properties of spines have not yet taken place, spines could play a significant electrical role, greatly influencing dendritic integration and the function of neural circuits.

Mechanisms of Calcium Decay Kinetics in Hippocampal Spines: Role of Spine Calcium Pumps and Calcium Diffusion through the Spine Neck in Biochemical Compartmentalization

Dendritic spines receive most excitatory inputs in the CNS and compartmentalize calcium. Although the mechanisms of calcium influx into spines have been explored, it is unknown what determines the calcium decay kinetics in spines. With two-photon microscopy we investigate action potential-induced calcium dynamics in spines from rat CA1 pyramidal neurons in slices. The [Ca(2+)](i) in most spines shows two decay kinetics: an initial fast component, during which [Ca(2+)](i) in spines decays to dendritic levels, followed by a slower decay phase in which the spine follows dendritic kinetics. The correlation between [Ca(2+)](i) in spine and dendrite at the breakpoint of the decay kinetics demonstrates diffusional equilibration between spine and dendrite during the slower component. To explain the faster initial decay, we rule out saturation or kinetic effects of endogenous or exogenous buffers and focus instead on (1) active calcium extrusion and (2) buffered diffusion of calcium from spine to dendrite. The presence of an undershoot in most spines indicates that extrusion mechanisms can be intrinsic to the spine. Supporting the two mechanisms, pharmacological blockade of smooth endoplasmic reticulum calcium (SERCA) pumps and the length of the spine neck affect spine decay kinetics. Using a mathematical model, we find that the contribution of calcium pumps and diffusion varies from spine to spine. We conclude that dendritic spines have calcium pumps and that their density and kinetics, together with the morphology of the spine neck, determine the time during which the spine compartmentalizes calcium.

Three-Dimensional Structure and Composition of CA3→CA1 Axons in Rat Hippocampal Slices: Implications for Presynaptic Connectivity and Compartmentalization

Physiological studies of CA3-->CA1 synaptic transmission and plasticity have revealed both pre- and postsynaptic effects. Understanding the extent to which individual presynaptic axonal boutons could provide local compartments for control of synaptic efficacy and microconnectivity requires knowledge of their three-dimensional morphology and composition. In hippocampal slices, serial electron microscopy was used to examine a nearly homogeneous population of CA3-->CA1 axons in the middle of stratum radiatum of area CA1. The locations of postsynaptic densities (PSDs), vesicles, and mitochondria were determined along 75 axon segments (9.1 +/- 2.0 micrometer in length). Synapses, defined by the colocalization of PSDs and vesicles, occurred on average at 2.7 micrometer intervals along the axons. Most varicosities (68%) had one PSD, 19% had 2-4 PSDs, and 13% had none. Synaptic vesicles occurred in 90% of the varicosities. One-half (53%) of the varicosities lacked mitochondria, raising questions about their regulation of ATP and Ca2+, and 8% of varicosities contained only mitochondria. Eleven axons were reconstructed fully. The varicosities were oblong and varied greatly in both length (1.1 +/- 0.7 micrometer) and volume (0.13 +/- 0.14 micrometer 3), whereas the intervaricosity shafts were narrow, tubular, and similar in diameter (0.17 +/- 0.04 micrometer) but variable in length (1.4 +/- 1.2 micrometer). The narrow axonal shafts resemble dendritic spine necks and thus could promote biochemical compartmentalization of individual axonal varicosities. The findings raise the intriguing possibility of localized differences in metabolism and connectivity among different axons, varicosities, and synapses.

EXPRESSION OF THE C4 PATTERN OF PHOTOSYNTHETIC ENZYME ACCUMULATION DURING LEAF DEVELOPMENT IN ATRIPLEX ROSEA (CHENOPODIACEAE)

Immunolocalization of the bundle sheath‐specific enzyme, ribulose‐1,5‐bisphosphate carboxylase/oxygenase (RuBPCase), and of the mesophyll‐specific enzyme, phosphoenolpyruvate carboxylase (PEPCase), was used to follow development of the C4 pattern of photosynthetic enzyme expression during leaf growth in Atriplex rosea. The leaf tissue used for this characterization was also used in a parallel ultrastructural study, so that the temporal coordination of developmental changes in enzyme expression and cell structure could be monitored. Bundle sheath‐specific accumulation of RuBPCase occurs early, at the time that bundle sheath tissue is delimited from the ground meristem, and follows the order of vein initiation. PEPCase proteins were detected 2–4 days after the first appearance of RuBPCase. PEPCase accumulation is restricted to ground meristem cells that are in direct contact with bundle sheath tissue and that will become C4 mesophyll; PEPCase was never found in more distant ground tissue. This pattern suggests that, while bundle sheath‐specific accumulation of RuBPCase coincides with formation of the appropriate precursor cells, PEPCase expression is delayed until mesophyll tissue reaches a critical developmental stage. Cell‐specific expression of both photosynthetic enzymes occurs well before the striking anatomical divergence of bundle sheath and mesophyll tissues, suggesting that biochemical compartmentation might serve as a developmental signal for subsequent structural differentiation.

Expression of the C 4 Pattern of Photosynthetic Enzyme Accumulation During Leaf Development in Atriplex rosea (Chenopodiaceae)

Immunolocalization of the bundle sheath-specific enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCase), and of the mesophyll-specific enzyme, phosphoenolpyruvate carboxylase (PEPCase), was used to follow development of the C4 pattern of photosynthetic enzyme expression during leaf growth in Atriplex rosea. The leaf tissue used for this characterization was also used in a parallel ultrastructural study, so that the temporal coordination of developmental changes in enzyme expression and cell structure could be monitored. Bundle sheath-specific accumulation of RuBPCase occurs early, at the time that bundle sheath tissue is delimited from the ground meristem, and follows the order of vein initiation. PEPCase proteins were detected 2–4 days after the first appearance of RuBPCase. PEPCase accumulation is restricted to ground meristem cells that are in direct contact with bundle sheath tissue and that will become C4 mesophyll; PEPCase was never found in more distant ground tissue. This pattern suggests that, while bundle sheath-specific accumulation of RuBPCase coincides with formation of the appropriate precursor cells, PEPCase expression is delayed until mesophyll tissue reaches a critical developmental stage. Cell-specific expression of both photosynthetic enzymes occurs well before the striking anatomical divergence of bundle sheath and mesophyll tissues, suggesting that biochemical compartmentation might serve as a developmental signal for subsequent structural differentiation.